Apparatus and methods for particle analysis and autofluorescence discrimination

ABSTRACT

Described herein are apparatuses and methods for analyzing an optical signal decay. In some embodiments, an apparatus includes: a source of a beam of pulsed optical energy; a sample holder configured to expose a sample to the beam; a detector comprising a number of spectral detection channels configured to convert the optical signals into respective electrical signals; and a signal processing module configured to perform a method. In some embodiments, the method includes: receiving the electrical signals from the detector; mathematically combining individual decay curves in the electrical signals into a supercurve, the supercurve comprising a number of components, each component having a time constant and a relative contribution to the supercurve; and quantifying a relative contribution of each component to the supercurve.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/128,943, entitled “Particle Analysis and Sorting Apparatus and Methods”, filed Dec. 22, 2020; and is a continuation-in-part of U.S. Nonprovisional patent application Ser. No. 17/209,057, entitled “Particle Analysis and Sorting Apparatus and Methods”, filed Mar. 22, 2021, the contents of each of which are herein incorporated by reference in their entirety.

This application claims the benefit of U.S. Provisional Patent Application Ser. No. 63/128,943, entitled “Particle Analysis and Sorting Apparatus and Methods”, filed Dec. 22, 2020; and is a continuation-in-part of U.S. Nonprovisional patent application Ser. No. 17/209,057, entitled “Particle Analysis and Sorting Apparatus and Methods”, filed Mar. 22, 2021, the contents of each of which are herein incorporated by reference in their entirety.

U.S. Nonprovisional patent application Ser. No. 17/209,057, filed Mar. 22, 2021 is a continuation of U.S. Nonprovisional patent application Ser. No. 16/725,332, entitled “Particle Analysis and Sorting Apparatus and Methods”, filed Dec. 23, 2019 and issued as U.S. Pat. No. 10,995,330 on Mar. 23, 2021; which is a continuation of U.S. Nonprovisional patent application Ser. No. 15/959,653, entitled “Particle Analysis and Sorting Apparatus and Methods, filed Apr. 23, 2018 and issued as U.S. Pat. No. 10,564,088 on Feb. 18, 2020, the contents of each of which are herein incorporated by reference in their entirety.

U.S. Nonprovisional patent application Ser. No. 15/959,653, filed Apr. 23, 2018 claims the priority benefit of U.S. Provisional Patent Application Ser. No. 62/593,995, entitled “Particle Analysis and Sorting Apparatus and Methods, filed Dec. 3, 2017, and is a continuation-in-part of U.S. Nonprovisional patent application Ser. No. 15/599,834, entitled “Particle Analysis and Sorting Apparatus and Methods,” filed May 19, 2017 and issued as U.S. Pat. No. 9,952,133 on Apr. 24, 2018, the contents of each of which are herein incorporated by reference in their entirety.

U.S. Nonprovisional patent application Ser. No. 15/599,834, filed May 19, 2017 is a continuation of U.S. Nonprovisional patent application Ser. No. 14/879,079, entitled “Particle Analysis and Sorting Apparatus and Methods,” filed Oct. 8, 2015 and issued as U.S. Pat. No. 9,658,148 on May 23, 2017; which claims the benefit of U.S. Provisional Patent Application Ser. No. 62/062,133, filed on Oct. 9, 2014, the contents of each of which are herein incorporated by reference in their entirety.

GOVERNMENT SUPPORT CLAUSE

This invention was made in part with government support under grant numbers 1R43GM123906-01, 1R43GM128546-01, 1R43GM131619-01 REVISED, 2R44GM123906-02A1, and 5R44GM123906-03 awarded by the National Institutes of Health. The government has certain rights in the invention.

INTRODUCTION

This disclosure pertains to the fields of Particle Analysis, Particle Sorting, Multiplexed Assays, Imaging, and Microscopy. In particular, embodiments disclosed herein are capable of increased multiplexing, reduced need for spectral compensation, and reduced interference from autofluorescence in Flow Cytometry, Cell Sorting, Bead-Based Multi-Analyte Assays, Imaging, and Microscopy.

BACKGROUND

Cellular analysis and sorting have reached a high level of sophistication, enabling their widespread use in life science research and medical diagnostics alike. Yet for all their remarkable success as technologies, much remains to be done in order to meet significant needs in terms of applications.

One area of continuing unmet need is that of multiplexing. Multiplexing (also referred to as multiparameter, high-parameter, multicolor, or polychromatic assays) refers to the practice of labeling cells, beads, or other particles with multiple types of biochemical or biophysical “tags” simultaneously and detecting those tags uniquely, so as to generate a richer set of information with each analysis. The most commonly used tags in microscopy and flow cytometry are fluorescent molecules, or fluorophores. A fluorophore may be a naturally occurring fluorophore; it may be an added reagent; it may be a fluorescent protein [like, e.g., Green Fluorescence Protein (GFP)] expressed by genetic manipulation; it may be a byproduct of chemical or biochemical reactions, etc. Fluorophores may be used as they are, relying on their native affinity for certain subcellular structures such as, e.g., DNA or RNA; or they may be linked to the highly specific biochemical entities known as antibodies, in a process referred to as conjugation. As a particular antibody binds to a matching antigen, often on the surface of a cell, the fluorophore conjugated to that antibody becomes a “tag” for that cell. The presence or absence of the fluorophore (and therefore of the antigen the fluorophore-conjugated antibody is intended to specifically bind to) can then be established by excitation of the cells in the sample by optical means and the detection (if present) of the fluorescence emission from the fluorophore. Fluorescence emission into a certain range of the optical spectrum, or band, is sometimes referred to in the art as a “color;” the ability to perform multiplexed analysis is therefore sometimes ranked by the number of simultaneous colors available for detection.

The use of multiple distinct tags (and detection of their associated colors) simultaneously allows the characterization of each cell to a much greater degree of detail than possible with the use of a single tag. In immunology particularly, cells are classified based on their expression of surface antigens. The identification of a large number of different surface antigens on various types of cells has motivated the creation of a rich taxonomy of cell types. To uniquely identify the exact type of cell under analysis, it is therefore often necessary to perform cell analysis protocols involving simultaneously a large number of distinct antibody-conjugated tags, each specifically designed to identify the presence of a particular type of antigen on the cell surface. Tags have also been devised to label intracellular components, such as certain proteins, and nucleic acids, in both single-stranded and double-stranded form.

In flow cytometry of the prior art, methods have been devised to simultaneously label cells with up to thirty or more different fluorescent tags and detect their respective colors. Commercially available instrumentation is generally limited to simultaneous detection of fifteen colors or less, and most commonly less than about ten colors. One of the main challenges of routinely performing highly multiplexed analysis (as the practice of simultaneously detecting more than about a dozen separate colors is sometimes called) is the technical difficulty of keeping detection of each color (and its associated tag) separate from detection of all the other ones. FIG. 1 illustrates one key aspect of the challenge of multiplexed measurement of fluorescence in the prior art. The graph in this FIG. 1 depicts various fluorescence emission curves (thin solid lines) of intensity (I) as a function of wavelength (A), all curves having been normalized to their respective peak intensities. In applications of fluorescence detection, it is very commonly desirable to employ several different colors, or spectral bands, of the electromagnetic spectrum, and to assign each band to a different fluorophore. Different fluorophores can be selected, on the basis of their average emission spectra, so as to obtain relatively dense coverage of a certain range of the electromagnetic spectrum, and thereby maximize the amount of information that can be extracted in the course of a single experiment or analysis “run.” However, when striving to maximize spectral coverage, one of the common undesirable consequences is spectral overlap. The shaded portions in FIG. 1 illustrate the problem caused by spectral overlap between adjacent fluorescence spectra. In this particular illustrative example, five spectral fluorescence “bands” or colors (the five emission curves peaking at different wavelengths) span a certain desired range of the electromagnetic spectrum, such as, e.g., the visible portion of the spectrum from about 400 nm to about 700 nm in wavelength. The shaded portions indicate sections of the spectrum where it is impossible, using spectral means alone, to decide whether the signal comes from one or the other of the two bands adjacent to the overlapping region; accordingly, the portions of the spectrum corresponding to significant overlap are commonly discarded, resulting in inefficient use of the spectrum. Additionally, even after discarding such portions, residual overlap remains in the other portions, resulting in contamination of one band from signals from other bands. Attempts at negating the deleterious effects of such contamination go under the heading of “compensation.” This spectral overlap problem is variously described in the literature and the community as the “crosstalk,” the “spillover,” the “compensation problem,” etc., and it is a major factor in limiting the maximum number of concurrent spectral bands, or colors, that can be employed in a fluorescence detection experiment.

Another potential problem in the spectral analysis of cellular samples is autofluorescence from endogenous fluorophores, i.e., fluorescence from molecular species already present in a cell prior to the addition of any external fluorescent labels. Separating or distinguishing autofluorescence from fluorescence due to exogenous biochemical or biophysical “tags” presents a challenge in many multiplexed analyses. Endogenous fluorophores may include: cyclic ring compounds (e.g., NADH, FAD), aromatic amino acids (e.g., tryptophan), cellular organelles (e.g., mitochondria, lysozyme), collagen and elastin, etc. For example, NADH (reduced form of nicotinamide adenine dinucleotide) and FAD (flavin adenine dinucleotide), two common metabolic cofactors found in living cells, absorb over a broad range of wavelengths, particularly in the ultraviolet (UV) (from about 200 to 400 nm) and blue-violet (400 to 500 nm) regions, and their autofluorescence overlaps with the visible emission spectra of commonly used fluorescent dyes.

It would be desirable, then, to provide a way to perform highly multiplexed analyses of particles or cells with a reduced or eliminated impact of spectral crosstalk and/or autofluorescence.

In cell analysis of the prior art, methods have also been devised to simultaneously label cells with up to thirty or more different tags and detect their respective characteristics. For example, the technique known in the art as mass cytometry employs not fluorescence as a way to distinguish different tags, but mass spectrometry, where the tags incorporate not fluorophores, but different isotopes of rare earths identifiable by their mass spectra. One major drawback of this approach is that the protocol of analysis is destructive to the sample, the cells and their tags becoming elementally vaporized in the process of generating the mass spectra. This approach is therefore not suited to the selection and sorting of cells or other particles following their identification by analysis.

It would be further desirable, then, to provide a way to perform selection and optionally sorting of particles or cells based on nondestructive highly multiplexed analysis with a reduced or eliminated impact of spectral crosstalk.

In bead-based multiplexing assays of the prior art, the substrate for the capture of analytes is the surface of a color-coded microsphere (also referred to as “bead”). The measurement of analytes (e.g., antigens) by so-called sandwich immunoassays is typically performed with, e.g., antigen-specific primary antibodies attached to the surface of the microsphere; the analytes are captured by the primary antibodies; and the reporting is typically performed using, e.g., secondary antibodies conjugated to fluorescent reporter molecules. Similar methods of the prior art are used for measurement of other analytes, including proteins, enzymes, hormones, drugs, nucleic acids, and other biological and synthetic molecules. To provide for simultaneous measurement of different analytes (multiplexing), each microsphere is internally stained with one or more dyes (colors) in precise amounts spanning a range of discrete levels. Each particular level of dye A (and optionally in combination with particular levels of dye B, dye C, etc.) is assigned to, e.g., a specific primary capture antibody attached to the surface of the microsphere. As color-coded beads are mixed with a sample, they each capture a certain analyte; a second step provides the secondary binding of the reporter molecule. The resulting bead+analytes+reporters complex is then passed through a particle analysis apparatus substantially very similar to a flow cytometer, where one light source is used to excite the dye or dyes in each bead, and another light source is used to excite the reporter fluorophore. The unique color code (combination of specific staining levels of dye A and optionally dye B, dye C, etc.) assigned to each capture entity allows the simultaneous analysis of tens or hundreds of analytes in a sample; the dye-based color coding of each bead is used to classify the results as the beads pass through. In current commercial offerings, there is a practical limit to the number of color-coded bead types that can be used simultaneously in a multiplex assay. One fluorescence detection spectral band is reserved for the reporter molecules, reducing the spectral range available for coding the beads; accordingly, it has been challenging to fashion more than two or three separate fluorescence detection bands out of the remaining available spectrum. Each band providing about 10 discrete levels of fluorescence for multiplexing, the total number of possible combinations is about 10 for one dye, about 100 for two dyes, and about 1,000 for three dyes. Current commercial offerings cap at 500 the number of practically available multiplexing combinations, limiting the number of individual analytes that can be examined in a single measurement run.

It would be further desirable, then, to provide a way to perform bead-based multiplexing with a greater number of simultaneously distinguishable beads, to enable the performance of multiplexing assays with a greater number of simultaneously measured analytes.

SUMMARY

One aspect of the present disclosure is directed to an apparatus for analyzing an optical signal decay. In some embodiments, the apparatus includes: a source of a beam of pulsed optical energy configured to expose a sample to the beam; a detector comprising a number of spectral detection channels sensitive to distinct wavelength sections of the electromagnetic spectrum, such that the channels are configured to detect optical signals resulting from interactions between the beam and the sample and convert the optical signals into respective electrical signals; a first optical path from the source of the beam to the sample; a second optical path from the sample to the detector; and a signal processing module configured to execute a lifetime analysis algorithm.

In some embodiments, the lifetime analysis algorithm comprises: receiving the electrical signals from the detector, such that the electrical signals represent a time-domain sequence of pulse signals; segmenting the sequence into equal pulse signal segments each comprising substantially a same number of sampling points, such that each sampling point of each pulse signal segment corresponds to a respective sampling index, and a length of each pulse signal segment corresponds to an excitation pulse repetition period; coherently adding a value of a sampling point corresponding to the respective sampling index of each pulse signal segment to form a supercurve, such that each sampling index of the supercurve corresponds to a value equal to a sum of substantially all the sampling point values from the corresponding sampling indices from each pulse signal segment, and the supercurve comprises an intensity of at least one lifetime component over time; determining whether the at least one lifetime component of the supercurve comprises one or both of: a short-lifetime component and a long-lifetime component; and quantifying an intensity contribution of the at least one lifetime component of the supercurve.

In some embodiments, the optical signals comprise a fluorescence signal.

In some embodiments, when present, the short-lifetime component comprises autofluorescence and the long-lifetime component comprises exogenous fluorescence.

In some embodiments, the at least one lifetime component comprises an instrument response function when the short-lifetime component and the long-lifetime component are not present.

In some embodiments, the sample comprises a suspension of particles, such that the apparatus further comprises: a flow path for the suspension of particles; and a flowcell configured as an optical excitation chamber for generating the optical signals from interactions between the beam of pulsed optical energy and the particles, such that the flowcell is connected with the flow path, the first optical path, and the second optical path.

In some embodiments, the apparatus further comprises: a particle sorting actuator connected with the flow path; an actuator driver connected with the actuator, such that the driver is configured to receive actuation signals from the signal processing module; and at least one particle collection receptacle connected with the flow path.

In some embodiments, the particle sorting actuator is based on at least one flow diversion in the flow path.

In some embodiments, the particle sorting actuator is based on one of: a transient bubble, a pressurizable chamber, a pressurizable/depressurizable chamber pair, or a pressure transducer.

In some embodiments, the signal processing module is further configured to extract time constants from the supercurve.

In some embodiments, the signal processing module is further configured to allocate each lifetime component of the supercurve to predetermined bins, each bin representing a group of relatively similar lifetime components.

Another embodiment of the present disclosure is directed to an apparatus for analyzing an optical signal decay. In some embodiments, the apparatus comprises a source of a beam of pulsed optical energy configured to expose a sample to the beam; a detector comprising a number of spectral detection channels sensitive to distinct wavelength sections of the electromagnetic spectrum, such that the channels are configured to detect optical signals resulting from interactions between the beam and the sample and convert the optical signals into respective electrical signals; a first optical path from the source of the beam to the sample; a second optical path from the sample to the detector; and a signal processing module configured to execute a lifetime analysis algorithm.

In some embodiments, the lifetime analysis algorithm comprises receiving the electrical signals from the detector, such that the electrical signals represent a time-domain sequence of pulse signals; segmenting the sequence into equal pulse signal segments each comprising substantially a same number of sampling points, such that each sampling point of each pulse signal segment corresponds to a respective sampling index, and a length of each pulse signal segment corresponds to an excitation pulse repetition period; coherently adding a value of a sampling point corresponding to the respective sampling index of each pulse signal segment to form a supercurve, such that each sampling index of the supercurve corresponds to a value equal to a sum of substantially all the sampling point values from the corresponding sampling indices from each pulse signal segment, and the supercurve comprises an intensity of at least one lifetime component over time; adding all the intensity values corresponding to each sampling index of the supercurve to generate a sum; normalizing the sum by dividing the sum by a peak value of the supercurve; determining a relative intensity contribution of each lifetime component by comparing the normalized sum to a lookup table; and determining an absolute intensity contribution of each lifetime component by multiplying the relative intensity contribution by the peak value of the supercurve.

In some embodiments, the signal processing module is further configured to subtract a baseline value from each respective sampling point.

In some embodiments, the signal processing module is further configured to measure a baseline shift produced by the at least one lifetime component as compared to a baseline when the at least one lifetime component is not present.

In some embodiments, the signal processing module is further configured to subtract the baseline shift from the received electrical signals.

Another aspect of the present disclosure is direct to an apparatus for analyzing an optical signal decay. In some embodiments, the apparatus comprises: a source of a beam of pulsed optical energy configured to expose a sample to the beam, such that the sample comprises one or more fluorophores having at least one lifetime component; a detector comprising a number of spectral detection channels sensitive to distinct wavelength sections of the electromagnetic spectrum, such that the channels are configured to detect optical signals resulting from interactions between the beam and the sample and convert the optical signals into respective electrical signals; a first optical path from the source of the beam to the sample; a second optical path from the sample to the detector; and a signal processing module configured to execute a lifetime analysis algorithm.

In some embodiments, the lifetime analysis algorithm comprises: receiving the electrical signals from the detector, such that the electrical signals represent a time-domain sequence of pulse signals; segmenting the sequence into equal pulse signal segments each comprising substantially a same number of sampling points, such that a length of each segment corresponds to an excitation pulse repetition period; measuring a peak baseline shift of the electrical signals based on a presence of the at least one lifetime component; and calculating an intensity contribution of the at least one lifetime component of the sample based on the measured peak baseline shift and one of: comparison to a lookup table and numerical fitting.

In some embodiments, the optical signals comprise a fluorescence signal.

In some embodiments, the at least one lifetime component is longer than the excitation pulse repetition period and comprises exogenous fluorescence.

In some embodiments, the signal processing module is further configured to subtract the baseline shift from the received electrical signals.

In some embodiments, the signal processing module is further configured to: coherently add a value of a sampling point corresponding to a respective sampling index of each pulse signal segment to form a supercurve, such that each sampling index of the supercurve corresponds to a value equal to a sum of substantially all the sampling point values from the corresponding sampling indices from each pulse signal segment, and the supercurve comprises an intensity of the at least one lifetime component over time; and quantifying an intensity contribution of the at least one lifetime component of the supercurve.

In some embodiments, the sample comprises a suspension of particles, such that the apparatus further comprises: a flow path for the suspension of particles; and a flowcell configured as an optical excitation chamber for generating the optical signals from interactions between the beam of pulsed optical energy and the particles; such that the flowcell is connected with the flow path, the first optical path, and the second optical path.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a wavelength diagram illustrating the spectral overlap (spillover) in multiplexing approaches of the prior art.

FIG. 2 is a time-domain diagram illustrating a frequency-based approach to measuring single-exponential fluorescence lifetime of the prior art.

FIG. 3 is a time-domain diagram illustrating a time-correlated single-photon counting approach to measuring fluorescence lifetime of the prior art.

FIG. 4A is a linear-linear time-domain diagram.

FIG. 4B is a log-linear time-domain diagram illustrating a single-exponential decay curve resulting from pulsed excitation.

FIG. 4C is a linear-linear time-domain diagram.

FIG. 4D is a log-linear time-domain diagram illustrating a double-exponential decay curve resulting from pulsed excitation.

FIG. 5 is a wavelength-lifetime diagram coupled with a wavelength diagram illustrating a multiplexing approach dense in both spectral bands and lifetime bins in accordance with one embodiment.

FIG. 6 is a wavelength-lifetime diagram coupled with a wavelength diagram illustrating a multiplexing approach sparse in both spectral bands and lifetime bins in accordance with one embodiment.

FIG. 7 is a schematic illustration of a system configuration of an apparatus for analysis of single particles in a sample in accordance with one embodiment.

FIG. 8 is a schematic illustration of a system configuration of an apparatus for analysis and sorting of single particles in a sample in accordance with one embodiment.

FIG. 9 is a schematic representation of the light collection and detection subsystem of a particle analyzer/sorter with a single spectral detection band in accordance with one embodiment.

FIG. 10 is a schematic representation of the light collection and detection subsystem of a particle analyzer/sorter with multiple spectral detection bands in accordance with one embodiment.

FIG. 11A is a time-domain diagram illustrating a signal processing sequence in accordance with one embodiment: interaction envelope due to a flowing particle crossing the beam.

FIG. 11B is a time-domain diagram illustrating a signal processing sequence in accordance with one embodiment: excitation pulses.

FIG. 11C is a time-domain diagram illustrating a signal processing sequence in accordance with one embodiment: effective excitation pulses.

FIG. 11D is a time-domain diagram illustrating a signal processing sequence in accordance with one embodiment: fluorescence emission pulses with decay curves.

FIG. 11E is a time-domain diagram illustrating a signal processing sequence in accordance with one embodiment: segmentation of individual pulse signals.

FIG. 11F is a time-domain diagram illustrating a signal processing sequence in accordance with one embodiment: construction of a supercurve.

FIG. 12A is a log-linear time-domain diagram illustrating a triple-exponential decay supercurve constructed from individual pulse signals resulting from pulsed excitation.

FIG. 12B is a log-linear time-domain diagram illustrating the process of computing successive index-pair differences, determining supercurve knee points, and determining supercurve time-constant branches.

FIG. 13A is a schematic plan-view illustration of one step, or state, of a particle analysis/sorting method that uses a sorting actuator in accordance with one embodiment.

FIG. 13B is a schematic plan-view illustration of one step, or state, of a particle analysis/sorting method that uses a sorting actuator in accordance with one embodiment.

FIG. 14A is a schematic cross-sectional illustration of one step, or state, of a particle analysis/sorting method with two sorting states and one-sided actuation in accordance with one embodiment.

FIG. 14B is a schematic cross-sectional illustration of one step, or state, of a particle analysis/sorting method with two sorting states and one-sided actuation in accordance with one embodiment.

FIG. 15A is a schematic cross-sectional illustration of one step, or state, of a particle analysis/sorting method with two sorting states and one-sided actuation in accordance with one embodiment.

FIG. 15B is a schematic cross-sectional illustration of one step, or states, of a particle analysis/sorting method with two sorting states and one-sided actuation in accordance with one embodiment.

FIG. 16A is a schematic cross-sectional illustration of one step, or state, of a particle analysis/sorting method with two sorting states and two-sided actuation in accordance with one embodiment.

FIG. 16B is a schematic cross-sectional illustration of one step, or state, of a particle analysis/sorting method with two sorting states and two-sided actuation in accordance with one embodiment.

FIG. 17A is a schematic cross-sectional illustration of one state of a particle analysis/sorting method with five sorting states and one-sided actuation that uses multiple sorting channels in accordance with one embodiment.

FIG. 17B is a schematic cross-sectional illustration of one state of a particle analysis/sorting method with five sorting states and one-sided actuation that uses multiple sorting channels in accordance with one embodiment.

FIG. 17C is a schematic cross-sectional illustration of one state of a particle analysis/sorting method with five sorting states and one-sided actuation that uses multiple sorting channels in accordance with one embodiment.

FIG. 17D is a schematic cross-sectional illustration of one state of a particle analysis/sorting method with five sorting states and one-sided actuation that uses multiple sorting channels in accordance with one embodiment.

FIG. 18A is a flow chart describing a sequence of principal operations involved in the performance of a method of lifetime analysis in accordance with one embodiment.

FIG. 18B is a flow chart describing a sequence of principal operations involved in the performance of a method of particle analysis in accordance with one embodiment.

FIG. 18C is a flow chart describing a sequence of principal operations involved in the performance of a method of highly multiplexed particle analysis in accordance with one embodiment.

FIG. 19 is a schematic representation of a data processing system to provide an analyzer/sorter in accordance with one embodiment.

FIG. 20A is a time-domain diagram illustrating computation of an area under the supercurve in accordance with one embodiment.

FIG. 20B is a time-domain diagram illustrating computation of an area under the supercurve in accordance with one embodiment.

FIG. 20C is a time-domain diagram illustrating computation of an area under the supercurve in accordance with one embodiment.

FIG. 21A illustrates an example of a fluorescence signal from a sample comprising one or more fluorophores whose longest fluorescence lifetime is substantially shorter than the interpulse period in accordance with one embodiment.

FIG. 21B illustrates an example of a fluorescence signal from a sample comprising one or more fluorophores, one of which has a fluorescence lifetime that is comparable to or longer than the interpulse period in accordance with one embodiment.

FIG. 22A is a flow cytometry histogram illustrating the high background attributable to autofluorescence in approaches of the prior art.

FIG. 22B is a flow cytometry histogram illustrating separation between unstained and stained (with an exogenous fluorophore) cellular populations by discriminating autofluorescence based on lifetime component analysis in accordance with one embodiment.

DETAILED DESCRIPTION

One possible solution to the spectral overlap problem in highly multiplexed particle and cell analysis would be to utilize, beside spectral information, another type of information with which to index or encode the tags used to label cell characteristics. By adding an independent quantity that can be detected and measured, one can significantly increase the number of combinations available to label and identify cell types. There would follow then a reduced need to fit a large number of independent spectral bands into a limited region of the electromagnetic spectrum, since the total number of available combinations could be allocated based on two independent quantities instead of just one.

One improvement disclosed herein is used to provide fluorescence lifetime as that independent quantity, to be combined with spectral labeling to generate a highly multiplexed set of independent combinations with which to uniquely tag different cell characteristics or cell types with a reduced or eliminated impact of spectral crosstalk.

Another improvement disclosed herein is used to provide the combination of fluorescence lifetime and spectral fluorescence labeling to aid not only in the highly multiplexed analysis of cells or other particles, but also in the selection and optional sorting of cells or other particles with a reduced or eliminated impact of spectral crosstalk.

Still another improvement disclosed herein is used to provide separation between autofluorescence and exogenous fluorescence using fluorescence lifetime as an independent quantity to aid not only in the highly multiplexed analysis of cells or other particles, but also in the selection and optional sorting of cells or other particles with a reduced or eliminated impact of background attributable to autofluorescence and optionally with a reduced or eliminated impact of spectral crosstalk from exogenous fluorophores.

Yet another improvement disclosed herein is used to provide the combination of fluorescence lifetime and spectral fluorescence labeling in bead-based multiplexing for antigen, protein, nucleic-acid, and other molecular assays. By providing for lifetime multiplexing of the dyes used to color code the beads, the present disclosure greatly expands the number of possible combinations that can be used to identify individual bead types. By adding the capability of distinguishing beads based on fluorescence lifetime binning, this number can be increased to 10,000, 100,000, or more, leading to orders-of-magnitude reductions in the cost of running, e.g., highly multiplexed immunoassays, protein assays, or nucleic-acid assays.

Fluorescence lifetime is an aspect of the fluorescence emission process governed by quantum-mechanical laws. Fluorescence is the absorption by an atom or molecule of a packet of optical energy (a photon) of a certain wavelength and the subsequent emission by the same atom or molecule of a packet of optical energy (another photon) at a longer wavelength. The amount of time elapsed between absorption and emission varies stochastically, but given an ensemble of isolated identical atoms or molecules, the frequency distribution of such elapsed times of the entire ensemble follows an exponential decay curve. The time constant of such a curve (the 1/e time) is referred to as the lifetime for that fluorescence transition for that atom or molecule.

Different molecular entities display different fluorescence transitions, characterized by different optimal wavelengths of optical absorption, different peak wavelengths of optical emission, and different fluorescence lifetimes. Certain molecular entities display fluorescence transitions with similar spectral characteristics (the profiles of emission as a function of wavelength schematically illustrated in FIG. 1 ) but with different fluorescence lifetimes. And other molecular entities display fluorescence transitions with different spectral characteristics but with similar fluorescence lifetimes. Accordingly, molecular entities may be selected based on spectral characteristics (spectral emission profile) and fluorescence lifetime as essentially independent quantities.

In order to use fluorescence lifetime as a multiplexing parameter in particle analysis, one needs to provide the means to measure it. FIG. 2 describes one aspect of measurements of fluorescence lifetime as carried out in one approach in the prior art. The graph in this FIG. 2 depicts two curves of optical intensity (I) as a function of time (t). In this approach, the intensity of optical excitation (thick solid line) is modulated at a certain frequency, and the resulting fluorescence signal (thin dashed line) is analyzed. The effect of a finite fluorescence lifetime manifests itself primarily in the phase shift between the modulated excitation and the modulated emission curves. The main drawback of this approach (so-called “phase-sensitive” or “frequency-domain” fluorescence lifetime) is that it can probe efficiently only one fluorescence lifetime component at a time, and is poorly suited to analysis of samples where more than one lifetime component should be measured simultaneously. Certain improvements disclosed herein are used to overcome this limitation.

FIG. 3 illustrates the principle behind another approach to measurement of fluorescence lifetime in the prior art. This approach has been referred to in the literature as Time-Correlated Single-Photon Counting (TCSPC), and has been used particularly in fluorescence lifetime imaging applications (FLIM). The graph depicted in this FIG. 3 shows two curves of intensity (I) as a function of time (t), both normalized to unit peak intensity, and a histogram with associated bins. The first curve (thick solid line) represents any one of many identical excitation pulses used to interrogate a portion of the sample; the second curve (thin dashed line) represents the inferred fluorescence emission response from the portion of the sample under interrogation. This second curve is not measured directly, but is instead inferred by a numerical fit to a histogram. A typical hypothetical histogram is shown as a series of boxes superimposed upon the second curve. This histogram represents the frequency distribution of arrival times of single fluorescence emission photons following excitation by a pulse. By exciting the same portion of the sample many times, a histogram is collected that faithfully reflects the underlying fluorescence decay curve. The main drawback of this approach is the very principle it is based on: single-photon counting. The method only works if a single photon is, on average, emitted as a result of excitation. For typical decay curves, it is not uncommon to require between tens of thousands and millions of repeated excitations in order to acquire enough statistics in the histogram for acceptable accuracy of results. Even at high pulse repetition rates, this approach necessarily results in dwell times (the time spent acquiring data on a single portion of the sample) on the order of milliseconds to seconds. Accordingly, TCSPC is an approach that has been successfully applied to stationary samples, but which is poorly suited to samples that are rapidly varying, unstable, flowing, or generally needing to be analyzed rapidly. Certain improvements disclosed herein are used to overcome this limitation.

FIGS. 4A-D illustrate the importance of direct time-domain measurements of fluorescence lifetime. In each of FIGS. 4A-D, a graph depicts the evolution in time (t) of the intensity (I) of two curves: the optical excitation pulse (shown as thick solid lines 410, 430, 450, and 470, in the four graphs, respectively) and the optical emission curve (shown as thin dashed lines 425, 445, 465, and 485, in the four graphs, respectively), both being normalized to unit peak intensity. In FIGS. 4A and 4C, each of the two curves in each graph 420 and 460 are plotted on a linear-linear scale; in FIGS. 4B and 4D, each of the two curves in each graph 440 and 480 are plotted on a log-linear scale (also known as a “semilog” scale). The graphs 420 and 440 in FIGS. 4A and 4B illustrate the same curves, just plotted on different scales; likewise, the graphs 460 and 480 in FIGS. 4C and 4D illustrate the same curves, just plotted on different scales. In fluorescence processes, a molecule (which can be naturally occurring, such as certain dyes, fluorescent proteins, or native cellular components such as NADH or FAD; induced by genetic manipulation, such as fluorescent proteins expressed by transfection; or chemically manufactured, such as a large portion of exogenous fluorophores in current use) exhibits a propensity for absorption of optical energy within a certain range of wavelengths (referred to as its absorption spectrum), followed by emission of optical energy into a different range of wavelengths (referred to as its emission spectrum). The process of absorption and emission in fluorescence is governed by quantum mechanics and is influenced by several factors. Some of those factors are intrinsic to the molecule; other factors are environmental factors. Emission of a fluorescent photon occurs stochastically; given a large enough collection of identical molecules (an ensemble), the collective emission of the ensemble will appear to decay over time. For a homogeneous ensemble (depicted in FIGS. 4A and 4B), the cumulative curve of fluorescence emission can be represented by a single decay lifetime (shown schematically as τ_(a) 421 in this case). In the semilog plot 440 of FIG. 4B, the single-decay nature of the dashed emission curve 445 is evidenced by the presence of a single straight slope (indicated by its corresponding lifetime τ_(a) 441, corresponding to the same τ_(a) as 421 in plot 420). For a heterogeneous ensemble (depicted in FIGS. 4C and 4D) consisting of, e.g., two distinct populations, the cumulative curve of fluorescence emission can be better represented by a compound function comprising two different decay lifetimes (shown schematically as τ_(b) 461 and τ_(c) 462 in this case). In the semilog plot 480 of FIG. 4D, the double-decay nature of the dashed emission curve 485 is evidenced by the presence of two straight-sloped branches (indicated by their respective lifetimes τ_(b) 481 and τ_(c) 482, respectively corresponding to τ_(b) 461 and τ_(c) 462 in plot 460) joined at a “knee.” The shape of the decay curve gives information regarding the environment of the molecules in the ensemble. Assuming that the ensemble in FIGS. 4C and 4D consisted of a single kind of molecular entity, the appearance of two distinct lifetimes might suggest that molecules in the ensemble are exposed to two different environmental influences, one of them causing a significant alteration to the native fluorescence lifetime of the molecules. Without a direct recording in real time of the actual shape of the emission decay curve of the ensemble of fluorescence molecules, the distinction between the cases depicted in FIGS. 4A-B and FIGS. 4C-D would be lost, and with it the information regarding the environment of the molecular species. The analysis made possible by this direct time-domain approach can be variously referred to as multi-component or multi-exponential fluorescence decay analysis.

A practical example of application of the principle of analysis of multi-exponential, or multi-component, fluorescence decay is found in the analysis of cells. A eukaryotic cell consists primarily of a membrane, a cytoplasm, a nucleus, and various subcellular cytoplasmic structures. The biochemical microenvironment experienced by a molecule within a cell is greatly affected by factors such as the local concentration of electrolytes, local pH, local temperature, etc. When a fluorophore enters a cell, its microenvironment may be very different, depending on whether the fluorophore is freely floating in the cytoplasm, binds to a molecule (e.g., RNA) or to an enzyme or other subcellular structures within the cytoplasm, or crosses the nuclear membrane to bind to, e.g., DNA in the nucleus. When exposed to optical excitation, the sub-ensemble of fluorophores bound to DNA in the nucleus may exhibit a very different lifetime from, e.g., the sub-ensemble of fluorophores freely floating in the cytoplasm. By analyzing the compound decay curve of the entire ensemble, one must be able to distinguish between the two (or more) different contributions to the lifetime, as an average single lifetime will blur the desired information and present an incomplete and/or misleading picture of the situation.

If the cell to be analyzed is stationary (as, e.g., adhered to a substrate, or grown on a substrate, suitable for placement under a microscope), existing microscopy tools could be used to spatially resolve physical locations within the cell, perform, e.g., highly repetitious experiments on single pixels (or voxels, as in certain applications of confocal scanning microscopy) spanning very small portions of the cell, and repeat these measurements over all pixels (or voxels, as in certain applications of confocal scanning microscopy) comprising the cell, e.g., in a two-dimensional array (or, in certain applications of confocal scanning microscopy, in a three-dimensional array), by raster scanning over or otherwise methodically interrogating the region of interest. There are however, many instances when that approach is not desirable, and it would be instead advantageous for the cell to be analyzed to be moving swiftly past the point of interrogation. One instance is when it is desirable to complete a set of measurements on a cell or on a group of cells in a very short time, as is generally the case in clinical diagnostic applications, where time-to-results is a critical parameter on which may depend patients' health or lives. A related instance, also of great relevance in clinical diagnostics and drug discovery and development, but increasingly also recognized as important in basic scientific research, is when it is desirable to complete a certain set of measurements on a very large collection of cells in a practical amount of time, so as to generate statistically relevant results not skewed by the impact of individual outliers. Another instance is when it is desirable to perform measurements on a cell in an environment that mimics to the greatest degree possible the environment of the cell in its native physiological state: As an example, for cells naturally suspended in flowing liquids, such as all blood cells, adhesion to a substrate is a very unnatural state that grossly interferes with their native configuration. There are yet instances where the details of the physical location within the cell (details afforded, at a price of both time and money, by high-resolution microscopy, including confocal scanning microscopy) are simply not important, but where a proxy for specific locations within the cell would suffice, given prior knowledge (based on prior offline studies or results from the literature) about the correlation between specific cell locations and values of the proxy measurement.

There are also instances where, regardless of the speed at which a measurement is carried out, and therefore regardless of whether the measurement is performed on a flow cytometer, under a microscope, or under some other yet experimental conditions, it would be desirable to reduce or eliminate the spectral crossover problem. Many analytical protocols in cell biology research, drug discovery, immunology research, and clinical diagnostics are predicated on the concurrent use of multiple fluorophores, in order to elucidate various properties of highly heterogeneous samples consisting of diverse cell populations, sometimes with uncertain origin or lineage. The spectral crossover inherent in such concurrent use of available fluorophores presently limits the multiplexing abilities of tools in current use—be they cell sorters, cell analyzers, image-based confocal scanning microscopes, or other platform. Various schemes have been developed to quantify and mitigate the deleterious impact of spectral crossover, and are generally referred to as compensation correction schemes. These schemes, however, suffer from overcomplexity, time burden, reagent cost burden, lack of reproducibility, and difficulty in the proper training of operators. It would be therefore advantageous to provide various analytical platforms with a way to multiplex complex measurements without the same attendant spectral crossover issue as is currently experienced.

There are yet instances of cellular analysis where it is desirable to perform fluorescence lifetime measurements on molecular species native to the systems under study. In this case the process of fluorescence is sometime referred to as autofluorescence or endogenous fluorescence, and it does not depend on the introduction of external fluorophores, but rather relies on the intrinsic fluorescence of molecules already present (generally naturally so) in the cell to be analyzed. Endogenous fluorescence is similar to the fluorescence of externally introduced fluorophores, in being subject to similar effects, such as the influence of the molecular microenvironment on fluorescence lifetime. Accordingly, it would be advantageous to be able to resolve different states of endogenous fluorescence on an analytical platform, so as to provide for simple and direct differentiation between cells belonging to different populations known to correlate with different values of endogenous fluorescence lifetime of one or more natively present compounds. One example of practical application of the principle of endogenous fluorescence is in the differential identification of cancer cells from normal cells based on metabolic information.

Several approaches of separating autofluorescence from exogenous fluorescence have been used in the prior art including, for example, avoiding specific fluorophores around autofluorescence-associated wavelengths, filter selection, gating-based strategies, and chemical quenching. However, these methods and many others require careful controls and complex experimental protocols, algorithms, and analysis schemes. In some instances, cellular autofluorescence has been eliminated using the extremely long lifetimes of europium chelates in microscopy or lanthanide chelates in flow cytometry. However, these methods are slow and poorly suited for anything but static or quasi-static imaging. One of the major advantages of flow cytometry is its extremely high throughput (often over 10,000 events/sec). Under flow conditions, each cell typically spends only a few microseconds in the measuring window. Adapting flow cytometry to measure fluorescence lifetime would require high laser modulation frequencies and high repetition rates to generate sufficient signal in such a short dwell time.

The present disclosure advantageously provides a solution for discriminating between autofluorescence and exogenous fluorescence. Separation of autofluorescence from exogenous fluorescence is another practical example of an application of multi-exponential, or multi-component, fluorescence decay analysis. Autofluorescent molecules in a cell versus biochemical tags attached to or introduced into a cell, when exposed to optical excitation, may each exhibit a very different lifetime but may overlap in emission spectra. By analyzing the compound decay curve of the entire ensemble, one must be able to distinguish between the two or more different contributions to the lifetime in any one spectral detection band. Returning to FIGS. 4C-4D, the two straight-sloped branches may represent, respectively, a short or autofluorescent lifetime component (lifetimes τ_(b) 481 corresponding to τ_(b) 461 in plot 460) and a long or exogenous fluorescence lifetime component (lifetime τ_(c) 482 corresponding to τ_(c) 462 in plot 460), joined at a transition or “knee”.

It will be appreciated by those skilled in the art that measurements of heterogeneous samples, whether biological or nonbiological, can comprise interrogation of different types of analytes (e.g., cells, particles, liquid mixtures, etc.). Any given sample, for example, may comprise cells with significant autofluorescence; cells without appreciable autofluorescence; and cells (whether or not autofluorescent) labeled or not labeled with different exogenous fluorescent tags. Accordingly, the decays observed in the course of sample interrogation may comprise multiple fluorescence lifetime components, a single fluorescence lifetime component, or may not comprise any fluorescence lifetime component at all. In this last case, the observed optical signal may be substantially null (or it may just comprise noise), but alternatively, it may display a pulse, residual from the excitation process due, e.g., to incomplete spectral filtering of the excitation light wavelength. This pulse may be referred to as the instrument response function (IRF) for a particular detection channel, since it is the pulse that results on that channel when no fluorescent species (whether autofluorescent or exogenously fluorescent) are present; generally such pulse will have a width that is limited by the response of the instrument, due to bandwidth limitations in the excitation drive electronics, in the excitation laser(s), in the detector(s), in the detector amplifier(s), in the digitizer(s), and/or in other electronic circuitry involved in the transduction of the optical signal, as well as to the shape of the optical excitation pulse itself. Typically the IRF pulse width is shorter than pulse widths resulting from fluorescent emissions. If such an IRF pulse is present, it can effectively behave as if it decays according to a characteristic lifetime, even though it comprises no fluorescence. Analysis of the signal can isolate this instance, for example by treating the IRF as a kind of “fluorescence” with its own lifetime value, which can be measured by observing unstained samples known to be non-autofluorescent. On channels where rejection of this residual by optical means is impractical, it is therefore possible to isolate the residual by treating it as a “fluorescent” species of its own. This allows the use of the same signal processing circuitry and algorithms used for fluorescence species identification, simply extended to include the possibility of a residual IRF pulse. Likewise, in a signal that does comprise actual fluorescence decay(s), the IRF can still be present, convolved with the fluorescence emission. By treating the IRF as an effectively (short-) lifetime fluorescent species, the present disclosure can isolate it from the desired fluorescent signals (whether from autofluorescence or exogenous fluorescence). As disclosed herein, a “lifetime component” may reflect a truly fluorescent species (whether autofluorescent or exogenously fluorescent) or, on certain detection channels, it may reflect the IRF residual.

FIG. 5 schematically illustrates a principle of the present disclosure, namely the ability to improve the multiplexing capacity of an analytical instrument by using fluorescence lifetime information. At the bottom of FIG. 5 is a graph 550 depicting various fluorescence emission curves 561-565 (thin solid lines) of intensity (I) as a function of wavelength (λ), all curves having been normalized to their respective unit peak intensity. In contrast to the prior art, where distinction between different fluorophores as endogenous fluorophores, cell labels, “tags”, or “markers” is performed exclusively by spectral means (the horizontal wavelength axis 2 in graph 550), in the present disclosure a separate, orthogonal dimension of analysis is added: the vertical lifetime r axis in the graph 500 at top. Each fluorescence emission curve is represented by a “band”, shown in the figure as a shaded vertical strip 511-515: FL₁ (511), FL₂ (512), FL₃ (513), FL₄ (514), FL₅ (515), . . . . Similarly, different fluorescence lifetime values are represented by different values of τ, grouped together in “bins”, shown in the figure as shaded horizontal strips 521-525: τ₁ (521), τ₂ (522), τ₃ (523), τ₄ (524), τ₅ (525), . . . . Each of the bins is intended to schematically represent a relatively similar group of lifetimes: the variation among the various lifetime values in the τ₁ bin will generally be smaller than the difference between the average lifetimes of the τ₁ and τ₂ bins, the variation among the various lifetime values in the τ₂ bin will generally be smaller than the difference between the average lifetimes of the τ₁ and τ₂ bins and will also generally be smaller than the difference between the average lifetimes of the τ₂ and τ₃ bins, and so on.

FIG. 5 makes it plain that the wavelength axis and its associated bands now represent only one dimension of a virtual plane. The second, added dimension of this virtual plane is represented by the fluorescence lifetime axis τ and its associated bins in graph 500. The schematic intersections of the wavelength bands and the lifetime bins are shown in graph 500 as darker shaded regions 530 (of which only a few are labeled) in the λ-τ plane. The increased multiplexing of the present disclosure is exemplified by the fact that, for every one of the spectral (wavelength) bands generally available to current analytical platforms, the present disclosure offers several possible multiplexed lifetimes: As an example, the fluorescence band FL₂ (512) supports multiple fluorescence lifetime bins τ₁ (521), τ₂ (522), τ₃ (523), τ₄ (524), τ₅ (525), . . . . For a system with n distinct fluorescence bands and m distinct lifetime bins, the total theoretical number of independent combinations is n×m; to use a practical example, for a system with 6 distinct fluorescence bands and 4 distinct lifetime bins, there are 6×4=24 mutually independent multiplexed combinations available.

FIG. 5 also describes how a specific example of a multiplexed combination would be resolved in the present disclosure. From the wavelength axis a particular spectral band (say, FL₃ band 513) is selected for analysis. This particular spectral band in practice would be selected by spectral optical means, such as one or more of thin-film filters, dichroic beam splitters, colored glass filters, diffraction gratings, and holographic gratings, or any other spectrally dispersing means suitable for the task and designed to pass this band of wavelengths preferentially over all others. The resulting optical signal could still comprise any of a number of fluorescence lifetimes, depending on the instrument design and on the nature of the sample. The spectral optical signal filtered through as FL₃ is detected, converted to electronic form, and sent to an electronic signal processing unit for digitization and further elaboration; see FIG. 10 . The signal processing unit (further described below in reference to FIGS. 7 and 19 ) performs an analysis of the decay characteristics of the optical signals corresponding to the particle under study, and allocates the various contributions to the overall signal from each possible band of lifetime values. A virtual electronic “bin” corresponding to the specific bin τ₄ (524) of lifetimes would receive a value corresponding to the fraction of the signal that could be ascribed as resulting from a lifetime decay within the acceptable range relevant for the τ₄ band. The combination of the spectral filtering for FL₃ performed optically on the emitted signal and the lifetime filtering for τ₄ performed digitally on the electronically converted optical signal results in narrowing down analysis to a single multiplexing element: the shaded intersection 540 marked with a thick solid square in graph 500 of FIG. 5 .

The specific choice of FL₃ and τ₄ is only illustrative, in the sense that any of the intersections between detectable spectral fluorescence bands and resolvable lifetime bins are potentially simultaneously accessible by analysis—resulting in the increased multiplexing ability described above as desirable. FIG. 5 shows explicitly a set of allowable multiplexed intersections for a prophetic example comprising 5 distinct fluorescence bands and 5 distinct lifetime bins: a resulting set of up to 5×5=25 separate, mutually independent combinations. This example is illustrative only: the number of possible combinations is not limited to 25, being given instead by the number of individually separable fluorescence spectral bands multiplied by the number of individually separable lifetime values bins. The theoretical maximum number of individually separable lifetime bins is related to the sampling frequency of digitization, the repetition rate of excitation pulses, and the duration (width) of each excitation pulse. In the limit of excitation pulses much shorter than lifetimes of interest (which are typically in the tens of picoseconds to tens of nanoseconds, and which in some cases reach microsecond levels or greater), the maximum number of separable lifetime bins is given by the pulse repetition period divided by twice the digitization sampling period, plus one. Electronic, optical, and other noise effects in actual systems may significantly reduce this theoretical maximum.

In the case where one of the lifetimes used is comparable to or substantially longer than the interpulse period (or an excitation pulse repetition period), which typically may be in the range of 5 to 500 ns, more preferably may be in the range of 10 to 200 ns, and most preferably may be in the range of 20 to 100 ns, the decay may be so comparatively slow as to produce an effectively flat optical emission baseline shift. One of skill in the art will appreciate that the terms interpulse period and excitation pulse repetition period may be used interchangeably herein. For example, fluorescence from common fluorophores typically has a longer lifetime (from about 4 ns or less to about 7 ns or more), while autofluorescence from cells has a predominantly short lifetime range, for example ranging from about 0.5 ns or less to about 3 ns or more. Fluorescence from certain nanocrystals (also referred to as inorganic nanoparticles or quantum dots) is longer still, ranging from about 15 or less to about 100 ns or more. For example, certain lanthanide complexes, including, without limitation, complexes of praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, and ytterbium, display long-lived fluorescence (also referred to as phosphorescence or luminescence) with lifetimes ranging from about 0.03 μs or less to about 1500 μs or more.

Measurement of the effective baseline shift produced by the long-lived fluorescent or luminescent compound, as compared to the baseline in the absence of the compound, allows the determination of the presence and, if present, of the amount of the long-lived compound. A long-lived compound can be used either in isolation or in addition to shorter-lived fluorescent species. FIGS. 21A and 21B illustrate two pedagogical examples of embodiments according to the present disclosure. In FIG. 21A, the sample under measurement comprises one or more fluorophores whose longest fluorescence lifetime τ₁ is substantially shorter than the interpulse period (or spacing or an excitation pulse repetition period) Δt₁. As a result, each of the interactions 2122 between an excitation laser pulse and the fluorophore(s) in the sample presents a fluorescence emission intensity I that rises to an approximate level I₁, then substantially decays to a zero baseline (represented here by the time abscissa t) before the next pulse. The fluorescence emission signal 2120 therefore comprises a series of approximately similar peaks spaced according to the interpulse period or an excitation pulse repetition period Δt₁. In one embodiment, factors affecting the shape and magnitude of signal 2120 are accounted for in a theoretical, mathematical, analytical, numerical, computational, and/or empirical model, and desired measurands (such as, without limitation, the fluorescence lifetime values of one or more fluorophores, the contributions of one or more fluorophores to the observed fluorescence emission intensity signal, and/or the number and concentration of fluorophores in the sample under interrogation) are extracted, e.g., by fitting to the observed signal, or quantities derived from it, a model in a process similar to that further described below.

In FIG. 21B, the sample under measurement comprises one or more fluorophores, one of which has a fluorescence lifetime Δt₂ which is comparable to or longer than the interpulse period (or spacing or an excitation pulse repetition period) Δt₂. For illustrative purposes, dotted curve 2132 indicates what the approximate shape of a single interaction between a laser pulse and the fluorophore(s) in the sample would be if the interpulse spacing were much longer than the fluorophore lifetime τ₂. With the interpulse spacing as indicated by Δt₂, the decay of fluorescence from the fluorophore(s) in the sample does not have enough time to reach zero baseline (represented here by the time abscissa t) before the next excitation pulse in the train arrives. As a result, the fluorescence emission signal 2130 builds up over time as a series of ratcheting or incremental steps 2134 over an increasing baseline 2136. Over the course of a sufficient number of excitation pulses (which number depends on factors including, without limitation, the intensity of the pulses, the duration of the pulses, the interpulse spacing, the number and concentration of absorbing fluorophores in the interrogation volume, and/or the fluorescence lifetime of the fluorophore), the baseline 2136 shifts asymptotically to a new steady-state level I₂. This baseline shift I₂ is measured and reported and/or used to calculate the number or concentration of the fluorophores present in the sample.

The pedagogical examples of FIGS. 21A and 21B are chosen to illustrate with clarity certain principles of the present disclosure. It will be apparent to those skilled in the art that a wide range of different configurations and outcomes are possible which are encompassed by this disclosure, even when they are not explicitly illustrated or described. For example, FIGS. 21A and 21B show schematically idealized fluorescence emission signals expected from certain limited intervals of interaction between an excitation pulse train and a sample comprising one or more fluorophores. FIG. 21A illustrates a portion of approximate fluorescence emission behavior resulting from a substantially constant interaction between the pulse train and, e.g., a particle in the sample. In the course of a typical such interaction, the magnitude of the emission generally rises and falls as the particle traverses the interrogating light beam, as shown schematically, e.g., by envelope 1115 in FIGS. 11A-11D. Over a suitably short period of time, particularly near the peak of the envelope, the magnitude of the interaction can be approximately constant as suggested by intensity I₁ in FIG. 21A, depending on factors such as, without limitation, the interpulse spacing, the beam waist dimensions, and/or the sample stream flow speed. Over the entire length course of the interaction, the shape of the interrogating beam profile generally causes the level I₁ in FIG. 21A to rise and fall similarly to envelope 1115 in FIGS. 11A-11D rather than remaining approximately constant.

Similarly, in FIG. 21B a portion of an idealized fluorescence emission signal resulting from an interaction between an excitation pulse train and, e.g., a particle in a sample is schematically illustrated for a case where the beam profile in the direction of particle and fluid flow comprises a rising-edge step function and a plateau. In other embodiments, the beam profile in the direction of particle and fluid flow follows a more gradual pattern, as shown schematically, e.g., by envelope 1115 in FIGS. 11A-11D. In such embodiments, the shape of the fluorescence emission signal 2130 generally rises and falls as the particle traverses the interrogating light beam, beginning with relatively smaller ratcheting or incremental steps 2134 that increase in magnitude as the envelope 1115 increases, reaching a peak (analogous to level I₂ in FIG. 21B), then decrease as the envelope 1115 decreases, eventually returning to the original baseline (represented here by the time abscissa t).

The change in baseline 2136 over time is generally the result of a number of factors, such as, without limitation, the intensity of the pulses, the duration of the pulses, the interpulse spacing (or an excitation pulse repetition period), the beam waist dimensions, the beam profile, the sample stream flow speed, the number and concentration of absorbing fluorophores in the interrogation volume, and/or the fluorescence lifetime of the fluorophores. As disclosed herein, the baseline shift may comprise a temporary plateau, as illustrated in FIG. 21B, or it may comprise an envelope with a well-defined peak, as discussed above. The terms “baseline shift” (i.e., the value of the plateau) and “peak baseline shift” (i.e., the value of the peak of the envelope) are therefore considered interchangeable within this context, indicating, in both cases, the maximum shift in baseline due to the presence of a fluorescent species having a lifetime comparable to or longer than the interpulse period. The baseline subtraction may be performed on the entire supercurve using an average or a peak value of the baseline shift, or it may be performed on a point-by-point basis, with each sampling point having an amount subtracted from it corresponding to the local value of the baseline shift.

The baseline shift measurement described herein can be combined with the fluorescence lifetime decay analysis also described herein. For example, a sample may comprise two or more fluorophores, one of which has a fluorescence lifetime τ₂ comparable to or longer than the interpulse spacing Δt₂, and another or other fluorophores which have fluorescence lifetimes shorter, or substantially shorter, than the interpulse spacing Δt₂. The fluorescence lifetime decay analysis described herein can in such cases be applied to the fluorescence decays in the ratcheting or incremental steps 2134. In one embodiment, the shifting baseline 2136, as described in connection to FIGS. 21A-21B, is subtracted from the measured fluorescence emission intensity signal 2130, and the fluorescence decay analysis described herein, e.g., in relationship to FIGS. 4A-D, 11A-11F, 12A-B, 18A-C, and 20A-C, is applied to the resulting baseline-subtracted signal. In another embodiment, factors affecting the shifting baseline 2136 are accounted for in a theoretical, mathematical, analytical, numerical, computational, and/or empirical model along with factors affecting fluorescence decay in the individual ratcheting or incremental steps 2134, and desired measurands (such as, without limitation, the fluorescence lifetime values of one or more fluorophores, the contributions of one or more fluorophores to the observed fluorescence emission intensity signal, and/or the number and concentration of fluorophores in the sample under interrogation) are extracted by fitting to the observed signal 2130, or quantities derived from it, the model in a process similar to that further described below. A longer-lived fluorophore responsible for the baseline shift described herein may be a known fluorophore, chosen by design and for which the disclosed apparatus and methods are optimized; or it may be an unknown fluorophore, the presence and amount (and optionally the identity) of which are detected by the disclosed apparatus and methods.

In practice it may be desirable to implement less than the theoretical maximum number of multiplexed combinations available by application of the present disclosure. Some of the practical reasons that may factor into the criteria for such a choice (which may be hard coded during design, or may alternately be left up to the instrument operator) may include: the desire to reduce the computational complexity required for a full implementation of the possible combinations; the desire to reduce the computational time required to perform a statistically acceptable analysis on the number of possible combinations; the desire to manufacture or to obtain a simpler, smaller, less costly instrument than would be needed for a full implementation of the theoretical maximum number of possible combinations; the desire for an operator to be able to operate the analytical platform with a minimum of specialized training; and the desire for a robust instrument designed to perform a reduced set of operations in a highly optimized fashion. Whichever the motivation, one may choose to produce a “sparse” multiplexed configuration, where some of the possible multiplexing choices have been removed.

In one embodiment of the present disclosure, such sparseness is introduced in the lifetime domain: Only a few of the possible lifetime bins are provided, the rest being removed and being replaced by gaps between the provided lifetime bins [e.g., removing bins τ₂ (522) and τ₄ (524) in FIG. 5 ]. The advantage of this configuration over a densely populated lifetime configuration is that the relative sparseness of the lifetime bins simplifies the process of digitally distinguishing the lifetime contributions of the remaining bins to the optical emission signal.

In another embodiment of the present disclosure, the sparseness of multiplexing is introduced in the spectral domain: Only a few of the possible wavelength bands are provided, the rest being removed and being replaced by gaps between the provided spectral bands [e.g., removing bands FL₂ (512) and FL₄ (514) in FIG. 5 ]. The advantage of this configuration over a densely populated spectral configuration is that the relative sparseness of the spectral bands simplifies the handling of any residual spectral overlap.

FIG. 6 shows an illustrative example (graph 600) of yet another embodiment of the present disclosure, where the sparseness of multiplexing has been introduced in both the spectral (graph 650 at bottom) and the lifetime domains simultaneously: Only a few of the possible wavelength bands have been provided, the rest having been removed and being indicated in the figure by the gaps between the provided spectral bands [e.g., between bands FL₁ (611) and FL₃ (613) and between bands FL₃ (613) and FL₅ (615)]; and only a few of the possible lifetime bins have been provided, the rest having been removed and being indicated in the figure by the gaps between the provided lifetime bins [e.g., between bins τ₁ (621) and τ₃ (623) and between bins τ₃ (623) and τ₅ (625)]. The resulting configuration, while having considerably fewer intersection points than the theoretical maximum, is however advantaged over a densely populated spectral and lifetime configuration by the reduction in the number of hardware components, the reduction in the complexity of the signal processing algorithms, by the relative increase in robustness and accuracy of the signal processing results, and by the relative simplicity of a training protocol for operation of the associated instrument platform.

FIG. 7 illustrates schematically a system configuration of an exemplary embodiment of the present disclosure, which provides an apparatus for highly multiplexed, compensation-reduced, and/or autofluorescence-interference-reduced particle analysis in a sample. In another embodiment, it provides an apparatus for lifetime analysis of particles in a sample. One or more light source 750, e.g., a laser, produces one or more optical energy (light) beams 722 with desired wavelength, power, dimensions, and cross-sectional characteristics. One or more modulation drivers 752 provide modulation signal(s) 702 for the one or more respective light sources, resulting in the beam(s) 722 becoming pulsed. The modulation drivers may optionally be internal to the light source(s). The pulsed beam(s) are directed to a set of relay optics 754 (which can include, without limitation, lenses, mirrors, prisms, and/or optical fibers), which may additionally optionally perform a beam-shaping function. Here relay optics will be intended to represent means to transmit one or more beams from one point in the system to another, and will also be intended to represent means to shape one or more beams in terms of dimensions and convergence, divergence, or collimation. The output pulsed beam(s) 732 from the beam-shaping relay optics are directed to another optional set of relay optics 758 (which can include, without limitation, lenses, mirrors, prisms, and/or optical fibers), which may additionally optionally perform a focusing function. The beam-shaping optics, the focusing optics, or both, may alternatively be incorporated into the light source module. The combined effect of the two sets of relay optics (the beam-shaping and the focusing sets) upon the input beam(s) from the light source(s) is to impart upon the beam(s) the desired output beam propagation characteristics suitable for interrogating particles, such as, without limitation, a desired focused beam waist, a desired ellipticity, a desired polarization, and/or desired wavefront characteristics, each on or about, e.g., the interrogation plane. The second set of relay optics then directs the pulsed beam(s) 708 to the flowcell 700. The flowcell 700 provides for the passage of particles to be analyzed (which can include, without limitation, cells, cell aggregates, bacteria, viruses, exosomes, liposomes, microvesicles, microparticles, nanoparticles, natural or synthetic microspheres, and chemical or biochemical molecules or compounds) by conveying a sample stream 740 containing said particles as a suspension, and a stream of sheath fluid 742 that surrounds and confines said sample stream, as further described herein. An input portion of the flowcell focuses, e.g., by hydrodynamic means, the sample stream and the surrounding sheath stream to result in a tight sample core stream flowing through a microchannel portion of the flowcell, surrounded by sheath fluid. The tight sample core stream flowing past the interrogation region of the flowcell typically exposes, on average, less than one particle at a time to the beam or beams for interrogation (this is sometimes referred to in the art as “single-file” particle interrogation even in those cases when the particles may not be substantially in single file, but may nevertheless generally pass one at a time through the interrogation region). The sheath fluid and the sample core stream are directed to a single outlet 744 (and generally discarded as waste) after passage through the interrogation portion of the flowcell. As the interrogating pulsed beam(s) of optical energy (light) interact with particles in the sample core stream by scattering, absorption, fluorescence, and other means, optical signals 710 are generated. These optical signals are collected by relay optics in box 760 (which can include, without limitation, single lenses, doublet lenses, multi-lens elements, mirrors, prisms, optical fibers, and/or waveguides) positioned around the flowcell, then conveyed to filtering optics in box 760 (which can include, without limitation, colored filters, dichroic filters, dichroic beamsplitters, bandpass filters, longpass filters, shortpass filters, multiband filters, diffraction gratings, prisms, and/or holographic optical elements) and then conveyed as filtered light signals 712 by further relay optics in box 760 to one or more detectors 770 (which can include, without limitation, photodiodes, avalanche photodiodes, photomultiplier tubes, silicon photomultipliers, avalanche photodiode microcell arrays, photodiode arrays, avalanche photodiode arrays, photomultiplier tube arrays, silicon photomultiplier arrays, and arrays of avalanche photodiode microcell arrays). The detectors convert the optical signals 712 into electronic signals 772, which are optionally further amplified and groomed to reduce the impact of unwanted noise. The electronic signals are sent to an electronic signal processing unit 790 [which generally comprises a digitization front end with an analog-to-digital converter for each signal stream, as well as discrete analog and digital filter units, and may comprise one or more of a Field-Programmable Gate Array (FPGA) chip or module; a Digital Signal Processing (DSP) chip or module; an Application-Specific Integrated Circuit (ASIC) chip or module; a single-core or multi-core Central Processing Unit (CPU); a microprocessor; a microcontroller; a standalone computer; and a remote processor located on a “digital cloud”-based server and accessed through data network or wired or cellular telephony means], which executes further processing steps upon the electronic signals. The processed signals 774 are then sent to a data storage unit 792 (which can include, without limitation, a read-only memory unit, a flash memory unit, a hard-disk drive, an optical storage unit, an external storage unit, or a remote or virtual storage unit connected to the instrument by means of a wired data or telecommunication network, a Wi-Fi link, an infrared communication link, and/or a cellular telephony network link). The stored or preliminarily processed data, or both, can also be made available to an operator for optional inspection of results.

FIG. 8 illustrates schematically a system configuration of an exemplary embodiment of the present disclosure, which provides an apparatus for highly multiplexed, compensation-reduced, and/or autofluorescence-interference-reduced analysis and sorting of particles in a sample. In another embodiment, it provides an apparatus for lifetime analysis and sorting of particles in a sample. It is similar in configuration to the system configuration of FIG. 7 , except in that it additionally provides for the capability to sort and collect particles based on their characteristics. The signal processing unit 890 generates in real time sorting control signals 876 based on the presence or absence or degree or nature of predetermined characteristics of the particles to be analyzed. For example, it may be desirable to identify and sort particles that, upon excitation by the interrogating pulsed light beam(s), emit fluorescence in a predefined spectral band at a level above a predefined threshold. As another example, it may be desirable to identify and sort particles that, upon excitation by the interrogating pulsed beam(s), exhibit fluorescence decay curve with a lifetime component in a certain range of values and at a percentage above a predefined threshold. In one embodiment, the predefined spectral band, range of lifetime values, and/or predefined threshold may correspond to a lifetime component attributable to autofluorescent molecules. Different criteria may be used in isolation or combined in compound logical forms (such as AND, OR, NOT, as well as more complex forms involving numerical comparisons of different quantities, such as, without limitation, “greater than,” “less than,” and so forth). The processing unit 890, once the processed signals from a given particle meet the predefined set of sorting criteria, triggers a signal 876 conveyed to an actuator driver 894. The actuator driver is an electronic control module connected to one or more sorting actuators 880. The sorting actuators may be positioned in, on, next to, or near the flowcell in the vicinity of, and downstream from, the interrogation region. One or more of the sorting actuators 880 is temporarily activated by drive signal 878 from the actuator driver 894 in response to the triggering signal 876 from the processing unit 890, resulting in a temporary diversion of the sample core stream, or of a portion of the sample core stream, away from the default (or waste) sorting channel 846 and into one or more sorting channels 848. The default sorting channel 846 optionally directs the fluids it receives into a default (or waste) receptacle 847. The sorting channel(s) 848 direct the sample core stream, in turn, to respective receiving sorting receptacle(s) 849. Once the temporary activation of one or more of the sorting actuators 880 is complete, the actuator(s) return to their resting state, and the sample core stream returns to its default (or waste) sorting channel 846. The sorting actuator(s) 880 are controllable to achieve multiple actuation states, including, without limitation, with an actuator driver 894, with a built-in control, with direct voltage or current control from the processing unit 890, or with electrical signals coming directly from logic circuitry connected with the one or more detectors 870.

In FIGS. 9 and 10 , the relative orientation of fluid flow, light propagation, and transverse directions is shown, respectively, as the set of axes x, z, and y. The process steps involved in the performance of some embodiments of the present disclosure are described here in reference to FIGS. 9 and 10 , and are also further summarized in flow-chart fashion in FIG. 18A.

FIG. 9 illustrates a cross-section, perpendicular to the direction of fluid flow, of a possible light collection configuration of the present disclosure. A flowcell 900, of which the inner part is schematically indicated in the figure, provides a channel for fluid flow. Sheath fluid 920 is provided to confine the fluid 930 carrying particles 955 to be analyzed. The sheath fluid and the sample-carrying fluid are focused into the flowcell lumen, optionally by hydrodynamic means; such focusing produces a tight sample core stream bounded by the sheath fluid. An interrogating light beam or beams 940 are provided to interact with the particles in the sample core stream. The beam or beams, usually having a Gaussian intensity profile, are generally focused into a relatively tight spot in the plane of the sample core stream. Particles to be analyzed in the sample core stream interact with light in the beam or beams 940 to generate optical signals 910 by optical processes including, for instance, scattering, absorption, or fluorescence. The optical signals 910 are collected by collection optics 960. The collected optical signals 912 are then conveyed (relayed) to spectral filtering optics 962 to select appropriate spectral bands of the optical signals for detection. The spectral filtering optics 962 may include, without limitation, reflective, transmissive, absorptive, diffractive, or holographic means, or means based on interference, or a combination thereof. The resulting spectrally filtered optical signals 914 are then conveyed (relayed) as signals 916 by focusing optics 964 to a detector 970. The detector converts the light signals 916 into electrical signals 972, which are then conveyed to a processing unit 990 for further analysis, processing, and optionally storage, as described above in reference to FIGS. 7 and 8 and as further discussed below. Together, the collection optics 960 and the focusing optics 964 may be referred to as relay optics.

In some embodiments, more than one spectral band output may be generated. For instance, FIG. 10 illustrates a cross-section, perpendicular to the direction of fluid flow, of another possible light collection configuration of the present disclosure. It is similar in concept to the configuration illustrated in FIG. 9 except that the spectral filtering optics 1062 produce more than one spectral band output 1014 (A and B), separated according to spectral characteristics. Each spectral band is then conveyed (relayed) to a separate set of focusing optics 1064 (A and B) and separate detectors 1070 (A and B), resulting in respectively separate electrically converted signals 1072 (A and B). The resulting electrical signals are then routed to signal processing unit 1090 for further elaboration. FIG. 10 depicts, for the sake of clarity, two sets of spectral bands, focusing optics, and detectors; it will be apparent to those skilled in the art that an arbitrary number of such sets is encompassed by the scope of the present disclosure.

The process steps described below in conjunction with FIGS. 11A-11F, 12A and 12B are also summarized in a flow-chart fashion in FIG. 18C.

FIGS. 11A-11F illustrate, for the specific case of implementation of the present disclosure on a flow cytometry platform, the principles involved in excitation, emission, detection, and analysis of fluorescence lifetime signals from particles under analysis. The various panels of the figure will be referred to in the text that follows to better illustrate the various steps of the signal transduction process. In each panel (A)-(F), a graph depicts the evolution over time (t) of certain optical intensities (I). In every case except where noted, the optical intensities are each shown as normalized to unit peak values for clarity of illustration; normalization of the optical intensities is preferable in certain embodiments, while in certain other embodiments the optical intensities are not normalized.

The graph 1110 in FIG. 11A depicts the canonical behavior of the optical signals resulting from the interaction between an always-on excitation light source (also referred to in the art as a constant-wave, or cw, source) and a particle passing through the region of interrogation (typically in a flow cell or other component having a similar function). As the particle enters, then exits, the region of interrogation, the excitation interaction signal (the dash-dotted line 1115) rises then falls, in concert with the spatial profile of the light beam used for interrogation, as measured along the line of passage of the particle. The particle, in this canonical pedagogical illustration, is assumed small in comparison with the dimension of the light beam along the direction of passage of the particle; modifications to this framework that generalize this to the case of particles of arbitrary size are possible but are not informative for the purpose at hand, and are not taken up here. For a typical light beam having dimensions, along the line of passage of particles to be analyzed, from less than 10 to more than 100 microns, and a typical flow cytometer causing particles to pass through the region of interrogation at flow velocities of below 0.1 to more than 10 m/s, the range of possible durations of the excitation interaction envelopes, as the shape of curve 1115 in FIG. 11A is sometimes referred to, is quite wide, stretching from less than 1 μs to more than 1 ms. However, in most cases in current practice the full-width at half-maximum (FWHM) of the excitation interaction envelope is from around a few microseconds to around a few tens of microseconds.

The graph 1120 in FIG. 11B juxtaposes, for illustrative purposes, the canonical interaction envelope 1115 from FIG. 11A (the dash-dotted line) with one possible configuration of excitation pulses from a modulated source of optical energy. The pulses are shown in a train of uniformly repeated, substantially identical units (the sharp features 1122 in thick solid lines); each pulse is short as compared to the FWHM of the canonical interaction envelope, and each pulse is separated from neighboring pulses by a time generally larger than the width of the pulses themselves. One key aspect of the present disclosure in FIG. 11B is that the modulation of the optical energy source (or sources) should result in a series of substantially identical pulses, each short compared to the typical interaction time, and each well separated from the next.

The graph 1130 in FIG. 11C depicts the prophetic result of delivering the train of excitation pulses 1125 illustrated in FIG. 11B to a particle flowing in a flow cell according to design and operating parameters typical of flow cytometer constructions known in the art. The resulting excitation interactions are shown as a series of pulses of varying height (features 1132 in thick solid lines) conforming to an overall envelope (the dash-dotted line), said envelope corresponding to the interaction envelope that would result, were the light beam continuous instead of pulsed and all other things remaining equal. While the details of the interaction sequence would, generally, vary from particle to particle (for example, the detailed timewise location of the individual interaction pulses 1132 in FIG. 11C under the overall envelope is a function of the relative timing of the pulse train with respect to the arrival of the particle), the general nature of the excitation interaction as consisting of a series of pulses modulated by a “carrier” envelope is determined by the design and operating parameters of the apparatus. In this graph 1130 of FIG. 11C, the individual interaction pulses are not normalized to unit intensity. And while in FIG. 11C are shown only six interaction pulses 1132 for clarity of illustration, it will be recognized by those skilled in the art that the present disclosure is not limited to such a number, the number of possible interaction pulses encompassed by the present disclosure being as small as one and as large as thousands or larger.

The graph 1140 in FIG. 11D adds another key element of the current disclosure to the picture, namely the ability to measure the temporal evolution of the fluorescence decay curves. The overall carrier envelope (dash-dotted line) and the individual excitation interaction pulses (features in thick solid lines) are as illustrated in FIG. 11C. The fluorescence decay curves are shown as thin dashed lines 1147. Each fluorescence decay curve follows directly the optical excitation associated with the interaction pulse immediately preceding it. It can be appreciated that the fluorescence decay curves are, generally, asymmetric: While the rising portion is dominated by the process of absorption of optical energy from the excitation source, the waning portion (the decay) is driven by the quantum mechanical processes of fluorescence emission, which vary from molecule to molecule and may additionally be affected by the molecular microenvironment, and generally result in a curve with a longer decay-side tail than the absorption-side tail. In this graph 1140 of FIG. 11D the individual interaction pulses and the individual fluorescence decay curves are not normalized to unit intensity.

The next process step in the lifetime analysis algorithm involves segmenting the signal sequence into individual decay curves. The dashed curves 1147 in graph 1140 of FIG. 11D represent optical emission signals, such as, e.g., fluorescence decay curves; these optical signals are detected by one or more detectors, converted into electrical signal, and digitized for further processing. In graph 1150 of FIG. 11E, the sequence of pulse signals (dashed curves, representing digitized electrical signals corresponding to the optical signals they are converted from) is broken (e.g., electronically, mathematically, numerically, digitally, computationally, or algorithmically) into individual pulse signal segments 1151-1156 (A, B, C, . . . ) while maintaining a consistent phase across the entire sequence; that is, a selected feature of each pulse (e.g., the peak, the midpoint of its rising edge, etc.) is chosen as the reference, and the sequence is cut up into equal segments (shown below the axis in FIG. 11E), all consisting of substantially the same number of digitization elements (also referred to as sampling points), and all starting substantially the same number L of digitization elements to the left of the respective reference point, such number L being chosen to result in segments, each of which (whenever possible) contains an entire decay curve not split between adjacent segments, as illustrated schematically in FIG. 11E. The segment length 1167 is chosen to closely match excitation pulse spacing 1166.

In an alternate embodiment, the reference points used to cut up the fluorescence signal sequence into substantially equal segments are drawn from a reference electronic signal derived from sources that include, without limitation: (i) the sequence of external pulses from a modulation device used to modulate the light source; (ii) a low-jitter, synchronized output function from the modulation device that has the same repetition frequency as the modulation pulse train; (iii) a low-jitter, synchronized output function from the internally modulated light source that has the same repetition frequency as the modulated pulse train; (iv) a low-jitter, synchronized output function from the externally modulated light source that has the same repetition frequency as the modulation pulse train; (v) the output of a fast photodetector designed to collect light from the excitation pulse train, e.g., as a “tap” on the main excitation beam, from surface scattering on, or partial reflections from, one or more optical components in the optical path of the excitation beam; and (vi) the output of a fast forward-scatter detector, a fast small-angle scatter detector, a fast intermediate-angle scatter detector, a fast side scatter detector, a fast backscatter detector, or generally any fast photodetector (including, without limitation, photomultiplier tubes, silicon photomultipliers, photodiodes, and avalanche photodiodes) arranged to detect excitation light scattered elastically by particles in the sample, and optionally designed to reject light inelastically scattered or converted by fluorescence processes; where “fast” means having the ability to convert detected light signals into electrical signals with sufficient bandwidth and at sufficient speed to minimize distortion, broadening, and other artifacts in the optoelectronic conversion process and, optionally, the preamplification and amplification processes. For example, for optical pulses between 0.5 and 10 nanoseconds in duration, it is preferable to have a bandwidth greater than 100 MHz, more preferable to have a bandwidth greater than 250 MHz, and most preferable to have a bandwidth greater than 2 GHz; other choices of bandwidth are also possible and may be desirable, depending on factors including, without limitation, component cost, the electronic noise of the detector/preamplifier at the chosen bandwidth, the degree of jitter present on the signal from other sources, and the electronic characteristics of other components in the signal detection/amplification/processing path. The reference electronic signal thus obtained provides a sequence of pulses, from each of which a selected feature (such as, e.g., the peak, the midpoint of the rising edge, etc.) is used as the reference for the demarcation of segment boundaries in the simultaneously collected fluorescence signal depicted in FIG. 11E.

Graph 1160 in FIG. 11F depicts the following step of signal processing by showing each of the decay curve segments 1151-1156 from FIG. 11E (A, B, C, . . . ) added coherently on top of each other, i.e., with the respective temporal relationships within each segment unchanged. Such adding is performed coherently on the basis of individual digitization elements. For example, each digitization index may be represented as an integer (e.g., 1, 2, 3, etc.). The values corresponding to the first digitization index (#1) in every segment are added together (A1+B1+C1 . . . ), the values corresponding to the second digitization index (#2) in every segment are added together (A2+B2+C2 . . . ), and so on for substantially all digitization indices in all segments. The result is a “supercurve” (bold curve 1165 in FIG. 11F), where each digitization index (e.g., #1, #2, #3, etc.) corresponds to a value that is equal to the sum of all the signal values (e.g., A1+B1+C1, A2+B2+C2, A3+B3+C3, etc.) from the corresponding digitization indices from all segments. The supercurve is then optionally converted to a semilog scale for further processing.

This signal processing method removes incoherent noise contributions from the result while boosting the contribution of signals coherent from pulse to pulse. The supercurve 1165 in graph 1160 of FIG. 11F may still exhibit some degree of incoherent noise, which is to be expected given the stochastic nature of the decay process and the presence of various sources of measurement noise on the signals; however, the general nature of the decay is expected to remain constant within a given population of fluorophores, and the supercurve process is aimed at maximizing the signal from such common decay while minimizing the effect of stray light signals, electronic noise, and other events lacking information content germane to the analysis being carried out.

FIGS. 12A and 12B illustrate exemplary embodiments of several steps of an analysis method of the current disclosure. Both FIGS. 12A and 12B display curves plotted on a semilog scale of the natural (or, alternatively, the base-10) logarithm axis of measured intensity vs. the linear axis of time. In graph 1250 of FIG. 12A the excitation pulse (bold solid curve 1201) and the resulting emission supercurve due, e.g., to fluorescence (dashed curve 1210) are each shown as normalized to unity peak value for clarity of illustration; on the shown logarithmic scale, a linear value of one corresponds to the logarithmic value of zero. Normalization of the emission supercurve is preferable in certain embodiments, while in certain other embodiments the supercurve is not normalized. The supercurve 1210 shown is obtained as described above in reference to FIGS. 11A-F for supercurve 1165. For illustrative purposes, the supercurve 1210 in graph 1250 of FIG. 12A is shown as comprising three distinct lifetime components [each also referred to herein as “component,” “lifetime,” “lifetime value,” “1/e value,” “time constant,” “decay constant,” or “exponential decay”, and corresponding to the value of τ in the standard exponential decay formula I(t)=I₀ exp(−t/τ), where I₀ is the starting intensity and I(t) is the intensity after a time t]: τ_(a), τ_(b), and τ_(c). In this example, τ_(a) is the smallest time constant of the three, m is the largest, and m is intermediate between the two. The relative values of τ_(a), τ_(b), and τ_(c) are reflected in the slopes of the three branches a (1221), b (1222), and c (1223) of the supercurve 1210: the slope of branch a 1221 is steepest, the slope of branch c 1223 is mildest, and the slope of branch b 1222 is intermediate between the two. The slope of a branch on a semilog plot of the kind depicted in FIGS. 12A and 12B is inversely proportional to the value of the corresponding time constant. The three branches 1221-1223 of the supercurve 1210 in FIG. 12A are defined as follows: The first branch a 1221 begins at or near the peak 1231 of the supercurve and ends at or near the first “knee” 1232 of the supercurve (where by “knee” is meant a substantial change in slope, indicated by an open circle); the second branch b 1222 begins at or near the first knee 1232 and ends at or near the next knee (the next open circle 1233); the third branch c 1223 begins at or near this next knee 1233 and ends at or near 1234 where the supercurve meets the measurement noise floor (schematically indicated in FIG. 12A by the time axis t). The slope of each branch is defined as customary as the ratio of the ordinate and the abscissa over a portion of or the entire branch: e.g., for branch b, the slope value is s_(b)=y_(b)/t_(b). The time constant corresponding to such slope is then obtained by the reciprocal of the slope: τ_(b)=1/s_(b).

It will be appreciated that when dealing with real measurements subject to effects including, without limitation, noise, background, uncertainty, instrument error or drift, component variability, and/or environmental effects, there may be departures, sometimes substantial, from the illustrations and depictions presented here. Even when such effects are low or minimized, other effects may act to mask, distort, alter, modify, or otherwise change the relationships among the various mathematical and physical quantities mentioned here. As one example, the noise floor where the supercurve in FIG. 12A starts and ends may be higher in certain cases and lower in others, depending on several factors, including, without limitation, the ones just mentioned. The variability in the noise floor may affect the determination of one or more of the slopes of the branches of the supercurve. Likewise, the precise location of a knee between two branches on the supercurve may be subject to uncertainty depending on the level of residual noise on the supercurve. Depending on the digitization sampling rate and on the noise present on the signal, the transition from one slope to the next (the knee) may occur more gradually than over a single data point, for example over two, three, or more data points. Another example of the distortion created by physical effects is shown in FIG. 12A where branch a 1221 is shown as beginning at the peak 1231 of the supercurve 1210, however the slope of this first branch does not immediately converge onto a stable value due to the roll-off from the peak. The degree of roll-off is dependent on the shape of the excitation pulse, the value of the first-branch lifetime, and other factors. These effects notwithstanding, one improvement of the present disclosure is used to minimize the impact of such effects. Construction of the supercurve from a number of individual pulse signals, with its attendant improvement in signal to noise, is one element that contributes to such minimization.

Another element is the relative simplicity in the extraction of desired parameters, such as the values of the time constants, from a supercurve. This is illustrated by graph 1280 in FIG. 12B. Graph 1280 shows a detail 1211 of the supercurve 1210 of FIG. 12A, where branch a 1281 (corresponding to branch a 1221 in graph 1250) ends and branch b 1282 (corresponding to branch b 1222 in graph 1250) begins. (The graph has been offset and rescaled in both abscissa and ordinate for illustrative purposes.) The two branches meet at the knee 1292 indicated by the open circle, corresponding to knee 1232 in graph 1250. Also plotted in FIG. 12B are the individual digitized points (also referred to as digital samplings) of the supercurve, indicated by small filled circles with solid drop lines to the time axis. The process step of determining the location of a knee (that is, the transition between one branch where a value of lifetime dominates, to another branch where a different value of lifetime dominates) comprises computing differences between successive values of the digitized supercurve. Four such difference for branch a are shown as δ_(a). Where residual noise on the supercurve is minimized, the value of δ_(a) from digitized point pair to digitized point pair will show little variation. Once the knee is crossed, however, the next computed difference will jump to δ_(b), and successive differences will once again remain substantially uniform around this new value. For one of the main improvements of the present disclosure, namely the provision of highly multiplexed means of particle analysis and sorting, it is not critical that the depicted successive values of δ_(a) be rigorously constant, nor those of δ_(b); it is merely sufficient that δ_(a) be different enough from δ_(b) to enable detection of the slope change at, or within a reasonably narrow range of, the indicated knee point. Sufficient difference between δ_(a) and δ_(b) is related to the precision and accuracy of the measurement system, the number, types, and severity of noise or error sources, and other factors. Detection of a discontinuous change in slope, however, is intrinsically simpler, instrumentally and computationally, than the absolute determination of the value of a slope.

Once a knee is found, the process continues until the entire supercurve is examined. The location of each knee, together with the location of the start of the first branch and the end of the last branch, define all the branches of the supercurve. The next processing step involves computing the average slope for each branch, which was described above in reference to FIG. 12A, and from such slope values the time constants of each branch are calculated. The following processing step involves allocating each branch to one of a set of predetermined lifetime (or time constant) bins. As illustrated in FIGS. 5 and 6 , one aspect of the present disclosure is the provision of a limited set of lifetime bins, where the lifetime within any one bin is allowed to vary somewhat, as long as the variation is not greater than the difference between neighboring bins. For the purpose of analyzing a supercurve and determining what lifetimes gave rise to the signals from which the supercurve was constructed, it is sufficient to establish (1) which of the lifetime bins was present in the measured particle or event (i.e., what fluorophores or other molecular species were present with a fluorescence decay value within the range of any one of the provided lifetime bins), and (2) the degree of relative contribution of each detected lifetime. For (1), the set of time constants computed from the branches of the supercurve as described above is compared to the set of allowed lifetime bins. In some cases there will be as many separate detected branches in a supercurve as there are bins: this would be the case for FIG. 12A, for example, if the number of allowed bins were 3. In other cases there will be fewer branches, indicating that a certain bin was not present (i.e., that no fluorophore or molecular species with a lifetime in the range of values of that bin was detected). By comparing the set of measured time constants with the set of allowed bins, a determination is made as to which bins are present in the measurement. Determination of the relative contribution of each detected lifetime (now associated with one of the allowed lifetime bins) is performed by comparing the values of the ordinates of each branch (the values y_(a), y_(b), and y_(c) in graph 1250 of FIG. 12A) with a calibration lookup table generated during manufacture of the apparatus. Such calibration lookup table is created by generating supercurves with known inputs, i.e., with 100% of one lifetime bin, 100% of another, and so on for all the lifetime bins selected to be available on the apparatus; then with varying mixtures of bins, such as, e.g., 10% of bin 1 and 90% of bin 2, 20% of bin 1 and 80% of bin 2, and so on until 90% of bin 1 and 10% of bin 2; repeating this for each pair of bins available on the apparatus. The resulting data provides the lookup table to compare measured lifetime ordinates (e.g., y_(a), y_(b), and y_(c)) with, and thereby determine, with interpolation if desired for greater accuracy, the relative contributions of each detected lifetime.

Another exemplary embodiment of the present disclosure involves performing curve fitting of a model (which can be, without limitation, mathematical, analytical, numerical, computational, empirical, or a combination thereof) to the constructed supercurve. The fitting procedure (using methods that include, without limitation, linear regression, nonlinear regression, least squares fitting, nonlinear least squares fitting, partial least squares fitting, weighted fitting, constrained fitting, Levenberg-Marquardt algorithm, Bayesian analysis, principal component analysis, cluster analysis, support vector machines, neural networks, machine learning, deep learning, and/or any of a number of other numerical optimization methods well known in the art) is designed to determine the most likely combination of lifetimes and contributions resulting in the observed supercurve. In the case of highly multiplexed analysis and sorting of particles based on lifetime analysis, an instrument is generally designed with a fixed number of known, discrete lifetime bins. Therefore, the fitting procedure does not require the determination of the lifetimes, but simply of the contributions of each lifetime component to the observed signal. This makes the fitting procedure much more constrained than would normally be the case, and this in turn makes the determination of lifetime contributions faster and computationally less expensive. In this exemplary embodiment, extraction of slopes from the supercurve, identification of each knee present in the supercurve, and determination of lifetime contributions from lookup tables are all replaced by the fitting procedure; the outcome of the fitting procedure is the best-fit set of contributions to the observed signals from each potentially contributing lifetime bin. In certain cases, the contributions from one, or more, or from all but one, or from all lifetime bins may be determined by fit to be zero or substantially zero; in certain cases, the fit may produce substantially nonzero contributions from one, from more than one, or from all lifetime bins. In the case of an apparatus for lifetime analysis of particles in a sample, where the lifetime or lifetimes are not known a priori, the fitting procedure would include determination of the lifetime values, as well as of the contributions of each lifetime to the observed signal.

Yet another embodiment of the present disclosure involves computing the area under the supercurve, and comparing the result with one or more lookup tables. FIGS. 20A-20C illustrate this procedure for three exemplary cases of a multiplexed system with two lifetime bins: (a) the case of a supercurve with only a short-lifetime (τ_(a)) decay, (b) the case of a supercurve with only a long-lifetime (τ_(b)) decay, and (c) the case of a supercurve with a two-lifetime (τ_(a) and τ_(b)) decay. Each of FIGS. 20A-20C displays curves plotted on a linear scale of measured intensity I vs. the linear axis of time t. Each graph 2050, 2051, and 2052 of FIGS. 20A-20C, respectively, shows the emission supercurve due, e.g., to fluorescence, as dashed curve 2010. The curves in these graphs are shown after baseline correction (which step is described below), and after normalization to unit peak height (which step is described below), for clarity of illustration; the raw measured intensity supercurves can generally take on a range of values of both baseline and peak height. The area under each curve is shown as the shaded regions 2035, 2036, and 2037 in FIGS. 20A, B, and C, respectively. In these Figures, the area is accumulated starting from the time 2031 when the supercurve 2010 reaches its peak value, and continuing through the rest of the supercurve. In an alternative embodiment, the entire area under the supercurve, including the unshaded area under the supercurve before the time 2031 when the supercurve reaches its peak value, is computed. In another embodiment, the area under the supercurve is computed starting from a time subsequent to the start of the supercurve but prior to the time 2031 when the supercurve 2010 reaches its peak value, and continuing through the rest of the supercurve. In yet another embodiment, the area under the supercurve is computed starting from a time subsequent to the time 2031 when the supercurve 2010 reaches its peak value, and continuing through the rest of the supercurve. In yet another embodiment, the area under the supercurve is computed starting from a time prior to or subsequent to the time 2031 when the supercurve 2010 reaches its peak value, and continuing to a second, later time prior to the end of the supercurve.

The area under the supercurve can be efficiently computed by sequentially adding successive measured intensity values of the supercurve, i.e., the values corresponding to exemplary digitized sampling points 2071 for the supercurve illustrated in FIG. 20A; the values corresponding to exemplary digitized sampling points 2073 for the supercurve illustrated in FIG. 20B; and the values corresponding to exemplary digitized sampling points 2075 for the supercurve illustrated in FIG. 20C. While only sets of three digitized sampling points are shown illustratively in each Figure, it will be readily apparent to someone of ordinary skill in the art that a supercurve may comprise many such points, as schematically indicated by the ellipses in each Figure, such as, e.g., tens, hundreds, or more points. To increase the accuracy of the value of the area under the supercurve, standard methods of numerical integration as are well known in the art may be optionally employed, including, without limitation, the trapezoidal rule, quadrature, splines, and interpolation. In an embodiment, the supercurve is not baseline-corrected, i.e., the sum of intensity values is not adjusted by subtracting a baseline. In another embodiment, the supercurve is baseline-corrected prior to the start of the area computation, i.e., a value corresponding to its baseline is subtracted from each measured intensity value to yield a supercurve with the corrected, zero-baseline 2038 illustrated in FIG. 20A. In another embodiment, the supercurve is baseline-corrected after adding the digitized sampling values together, i.e., a value corresponding to its baseline multiplied by the number of added digitized sampling values is subtracted from the sum of intensity values to yield a supercurve with the corrected, zero-baseline 2038 illustrated in FIG. 20A. Baseline correction is performed using any of a number of methods well known in the art, including filtering, signal conditioning, analog signal processing, digital signal processing, and hybrid signal processing methods. In an embodiment, the sum of intensity values (whether baseline-corrected or not) is then optionally multiplied by the sampling interval 2039 (δt) to yield the areas 2035, 2036, and 2037, respectively, in each of the exemplary cases illustrated in FIGS. 20A, B, and, C. In another embodiment, for greater computational efficiency, this step can be skipped, as long as the values in the lookup tables that the measured area is compared to are generated in an analogous way; to avoid confusion, in what follows we will refer to “area under the supercurve” (alternatively, “supercurve area”) interchangeably as either the sum of intensity values, or such sum multiplied by the sampling interval 2039. The computed area under the supercurve is then normalized by dividing it by the raw measured peak value reached at time 2031.

The computed normalized value of the area under the supercurve is then compared with values in a lookup table constructed to cover a specified range of possible supercurve area values that can be obtained in a measurement. Such a lookup table includes relatively low values of the area, such as area 2035 depicted in FIG. 20A; relatively high values of the area, such as area 2036 depicted in FIG. 20B; and relatively intermediate values of the area, such as area 2037 depicted in FIG. 20C. For the exemplary case illustrated here of a multiplexed system with two lifetime bins, the lookup table furnishes, for each value of supercurve area, a corresponding pair of values: (i) the relative contribution of the short-lifetime (Ta) component of decay, and (ii) the relative contribution of the long-lifetime (m) component. In the case illustrated in FIG. 20A, the lookup table would provide a relative contribution of the short-lifetime component of 100% or approximately 100%, and a relative contribution of the long-lifetime component of 0% or approximately 0%; in the case illustrated in FIG. 20B, the lookup table would provide a relative contribution of the short-lifetime component of 0% or approximately 0%, and a relative contribution of the long-lifetime component of 100% or approximately 100%; in the case schematically illustrated in FIG. 20C, the lookup table would provide a relative contribution of the short-lifetime component of 50% or approximately 50%, and a relative contribution of the long-lifetime component of 50% or approximately 50%. The actual values of each relative contribution are provided in the lookup table previously generated in a calibration process, where reference supercurve area values are obtained from sets of samples with only a short-lifetime component, only a long-lifetime component, and known mixtures of both components in varying ratios, from ratios approaching pure short-lifetime component (such as, e.g., 3,000:1, 10,000:1, or higher) to ratios approaching pure long-lifetime component (such as, e.g., 1:3,000, 1:10,000, or higher), through intermediate ratios (such as, e.g., 1,000:1, 300:1, 100:1, 30:1, 10:1, 3:1, 1:1, 1:3, 1:10, 1:30, 1:100, 1:300, and 1:1,000). A lookup table may be provided with relatively few, such as, e.g., 10 or less, values of supercurve area; relatively many, such as, e.g., 1,000,000 or more, values of supercurve area; or a relatively intermediate number, such as, e.g., 100, 1,000, 10,000, or 100,000, values of supercurve area.

The process of comparing the measured supercurve area with values in the lookup table may comprise finding the closest value in the table, interpolating linearly between the two closest values, interpolating quadratically between the two closest values, extrapolating beyond the first or last value linearly, quadratically, or based on higher-order polynomial or on exponential curves, or any number of additional numerical estimation processes well known in the art. Once the closest value in the lookup table (or an interpolated or extrapolated value) is found, the corresponding pair of relative contributions (or the corresponding pair of interpolated or extrapolated relative contributions) of each lifetime component is obtained. Each relative contribution is then multiplied by the peak unnormalized intensity value of the supercurve reached at time 2031 in order to obtain the absolute intensity contributions of each lifetime component to the measured signal (these values are equivalent to ones known in the art as “height” or “peak” signal values). Each relative contribution is optionally multiplied by a factor S in order to obtain the absolute area contributions of each lifetime component to the measured signal (these values are equivalent to ones known in the art as “area” signal values): the factor S is calculated by adding the peak values of the raw signal pulses (i.e., pulses 1151, 1152, . . . , 1156 in FIG. 11E) and multiplying the result by pulse-to-pulse spacing 1166. To increase the accuracy of computation of the “area” signal values, standard methods of numerical integration as are well known in the art may be optionally employed in determining factor 5, including, without limitation, the trapezoidal rule, quadrature, splines, and interpolation. For the avoidance of confusion, the “area” signal values referred to here refer to an estimation of the area under the total envelope 1115 of interaction between a particle and the light beam in FIG. 11A, whereas the “area under the supercurve” refers to an estimation of the area under supercurve 1165 in FIG. 11F.

While certain embodiments described herein used an exemplary configuration of a multiplexing system with two lifetime bins, it will be readily apparent to someone of skill in the art that other configurations, including ones with three, four, or more lifetime bins, are encompassed by this disclosure, an example of which is described in connection with FIGS. 21A-21B. While the illustrative exemplar described herein is based on representing and operating on supercurves, summed intensity values, and areas on linear scales, alternative embodiments comprise representing and operating on supercurves, summed intensity values, and areas on logarithmic or other scales. It will also be readily apparent to someone of skill in the art that while a specific example is given of a method involving a lookup table generated with two specific lifetime components (τ_(a) and τ_(b)) in varying ratios, a system of the current disclosure may also be provided with two, three, or more separate lookup tables, each table referring to a combination of different lifetimes (including, without limitation, for the case of two lifetimes: a common short lifetime τ_(a) and a different long lifetime τ_(c); a common long lifetime τ_(b) and a different short lifetime τ_(d); a different short lifetime τ_(e) and a different long lifetime τ_(f), and additional similar such combinations). The system may be provided with an option for the user to select a certain combination of lifetimes based on the type of assay being performed, or the system may be provided with a fixed configuration of certain lifetimes. Furthermore, each detection channel may be provided with the same lookup table, or each detection channel may be provided with a different lookup table (or a set of lookup tables) based on the combination of lifetimes assigned by default to, or selectable by the user for, that channel.

FIGS. 22A and 22B illustrate exemplary flow cytometry fluorescence histograms in populations of eosinophils (indicated as “Eos” in FIGS. 22A and 22B), a granulocytic subset of white blood cells known in the art to exhibit relatively high levels of autofluorescence. FIG. 22A, a representative diagram illustrating limitations of the prior art, shows histogram distributions of flow cytometrically analyzed stained cell population 2220 (“stained Eos”) and unstained cell population 2210 (“unstained Eos”). Both populations are interrogated with laser light at 375 nm, and emitted light is collected, spectrally filtered into a band ranging from approximately 442 nm to approximately 478 nm, and detected by a photodetector. The stained population is labeled with the conjugate CD45/AlexaFluor405; the observed signal comprises both signal from bound CD45/AlexaFluor405 (exogenous fluorescence) and cellular autofluorescence from the eosinophils themselves (endogenous fluorescence). The unstained population is not labeled, and therefore presents no fluorescence except its own cellular autofluorescence. As FIG. 22A shows, the two populations overlap nearly completely due to the relatively elevated levels of autofluorescence in the samples. In other words, the system of the prior art is unable to effectively discriminate autofluorescence from exogenous fluorescence in the detected signal, drastically reducing assay sensitivity. This makes it difficult to analyze eosinophil populations in the prior art.

By contrast, FIG. 22B, a representative diagram illustrating improvements of the present disclosure, shows histogram distributions of stained cell population 2225 (“stained Eos”) and unstained cell population 2215 (“unstained Eos”) analyzed on one flow cytometric embodiment of the present disclosure. The samples, labeling, excitation, and detection parameters are similar to those in FIG. 22A, except that in FIG. 22B hardware configuration and lifetime component analysis according to the present disclosure were used to discriminate, separate, and subtract a short-lifetime component, attributable to cellular autofluorescence, from a long-lifetime component, attributable to exogenous fluorescence. As a result, the unstained population 2215, thereby removed of interference from autofluorescence, becomes well separated from and no longer overlaps with the stained population 2225 (likewise removed of interference from autofluorescence), significantly improving the ability of the apparatus and methods of the present disclosure to analyze cellular populations.

FIGS. 13A-13B illustrate exemplary embodiments of two steps of an analysis and sorting method of the current disclosure. In FIGS. 13A-13B, the relative orientation of fluid flow, light propagation, and transverse directions is shown as the set of axes x, z, and y, respectively. The assignment of the axes and directions is similar to that in FIGS. 9 and 10 , however the orientation of the axes with respect to the page is rotated as compared to FIGS. 9 and 10 , with the light propagation and flow directions being in the plane of the page in FIGS. 13A and 13B. Each of the two figures shows a schematic representation of a side view of the interrogation region 1331 and sorting region 1332 of the flowcell 1300. The focusing region of the flowcell, if provided, e.g., by hydrodynamic means, is to the left of the picture; the sample core stream 1330, surrounded by the sheath fluid 1320, comes in from the left and flows towards the right. The sheath fluid 1320 is bounded by the inner walls of the flowcell 1300, and the sample core stream 1330 is bounded by the sheath fluid 1320. In the interrogation region 1331 at left, one or more beams of pulsed optical energy 1340 are delivered to the flowcell by relay/focusing optics and intersect the sample core stream 1330. In the sorting region 1332 at right, one or more actuators (shown in the picture as actuator 1380) are provided in contact with or near the flowcell, positioned in such a way as to overlay the position of the sample core stream 1330.

FIG. 13A shows a first time step in the processing of a sample whereby a single particle 1355 in the sample core stream 1330 enters the interrogation region 1331 (where the beam or beams 1340 intersect the sample core stream 1330). The light-particle interaction generates light signals as described above in reference to FIG. 9 or 10 , which light signals are collected and relayed to one or more detectors. The detector(s) record the optical interaction signals generated by the particle 1355, and convey that information to the signal processing unit as illustrated schematically in FIG. 8 . As described above in reference to FIG. 8 , the processing unit uses that information to produce, if certain predetermined criteria are met, a triggering signal for an actuator driver, which driver in turn activates the actuator 1380 in FIG. 13A. FIG. 13B shows a second time step in the processing of the sample whereby the particle 1355 detected in the step depicted in FIG. 13A, after following path 1365 in the flowcell along direction x, arrives at a point in the vicinity of the actuator 1380 in the sorting region 1332 of the flowcell. The design of the flowcell, of the optical layout of the actuator, and of the detection, processing, and control electronics is such that the actuator is activated as such a time when the passing particle is calculated, estimated, predicted, or found, upon calibration or determined empirically during instrument design or assembly, to be nearest to a position where activation of the actuator results in the desired diversion of the core stream to one of the one or more sorting channels. The timing of the triggering signal (i.e., the relative delay from particle detection to sorting actuation) is designed to take into account both the average velocity of fluid flow in the flowcell and its spatial profile across the flowcell cross-section, according to the characteristics of Poiseuille flow known in the art and as modified based on empirical or modeling information.

In FIGS. 14A and B, 15A and B, 16A and B, and 17A-17D, the relative orientation of fluid flow, light propagation, and transverse directions is shown as the set of axes x, z, and y, respectively. The assignment of the axes and directions is similar to that in FIGS. 9 and 10 , however the orientation of the axes with respect to the page is rotated as compared to FIGS. 9 and 10 , with the fluid flow and transverse directions being in the plane of the page in FIGS. 14A and B, 15A and B, 16A and B, and 17A-17D. The cross-sectional plane depicted in FIGS. 14A and B, 15A and B, 16A and B, and 17A-17D is the plane that contains the sample core stream.

FIGS. 14A and 14B illustrate one embodiment of two states of the particle analysis and sorting method of the current disclosure. Each of the two figures shows a schematic representation of a cross-sectional view of the sorting region of the flowcell. Similarly to the situation depicted in FIGS. 13A and 13B, the focusing region of the flowcell, e.g., by hydrodynamic means, if provided, is to the left of the picture; the sample core stream 1430, surrounded by the sheath fluid 1420, comes in from the left and flows towards the right. The sheath fluid 1420 is bounded by the inner walls of the flowcell 1400, and the sample core stream 1430 is bounded by the sheath fluid 1420. The flowcell 1400 splits into two channels in the sorting region: the default (or waste) sorting channel 1446 and the sorting channel 1448. Actuator 1480 is depicted as embodied in, in contact with, or in proximity of the inner wall of the flowcell 1400 on the default sorting channel side. FIG. 14A shows the configuration of the default state, where with the actuator 1480 in the OFF state, a non-selected particle 1450 in the sample core stream 1430 flows by design into the default sorting channel 1446. Similarly to the state depicted in FIG. 13B, FIG. 14B shows the configuration of the sorting state, where with the actuator 1480 in the ON state, a transient gas, vapor, or gas-vapor bubble, or a region of heated or cooled, less-dense sheath fluid 1495 is generated (by means including, without limitation, thermal means, electrolytic means, and/or gas injection means), which creates a localized flow diversion in the depicted cross-sectional plane and in its vicinity, which diversion temporarily deflects the sample core stream 1431 into the sorting channel 1448, which sample core stream contains a particle 1455 detected upstream and automatically selected by analysis circuitry and/or algorithms to trigger sorting actuation. Following deactivation of the actuator 1480, the transient gas, vapor, gas-vapor bubble or region of less-dense fluid 1495 shrinks or is cleared away, and the flow pattern returns to the original default state of FIG. 14A.

FIGS. 15A and 15B illustrate another embodiment of two states of a particle analysis and sorting method of the current disclosure. It is similar to the embodiment illustrated in FIGS. 14A and 14B, except in the design and nature of actuation. Here the actuator 1580 is located in proximity to an expandable chamber 1597 adjacent to the flowcell inner wall and separated from the sheath fluid 1520 by a flexible membrane 1596. With the actuator 1580 in the OFF or default state as shown in FIG. 15A, the expandable chamber 1597 is in its default configuration at a pressure designed to match the pressure of the fluid inside the flowcell at the location of the membrane, resulting in a flat shape of the membrane to match the shape of the flowcell inner wall, and a non-selected particle 1550 in the sample core stream 1530 flows by design into the default (or waste) sorting channel 1546. With the actuator 1580 in the ON or sorting state as shown in FIG. 15B, the expandable chamber 1597 is pressurized (by means including, without limitation, thermal means, mechanical means, hydraulic and/or gas injection means) to a higher pressure than in the default configuration; this pressure differential causes the membrane 1596 to flex into the flowcell until a new equilibrium is reached. The bulging membrane causes the flow pattern to shift in a similar way to that previously shown for FIG. 14B, resulting in the sample core stream 1531 being temporarily diverted into the sorting channel 1548, which sample stream contains a particle 1555 detected upstream and automatically selected by analysis circuitry and/or algorithms to trigger sorting actuation. Following deactivation of the actuator 1580, the expandable chamber 1597 is allowed to or made to return to its default pressure state, the membrane 1596 returns to its default flat shape, and the flow pattern returns to the original default configuration of FIG. 15A.

FIGS. 16A and 16B illustrate yet another embodiment of two states of a particle analysis and sorting method of the current disclosure. It is similar to the embodiment illustrated in FIGS. 15A and 15B, except in the design of actuation. Sorting actuation here is realized by means of two actuators, positioned on opposite sides of the flowcell, each actuator being located in proximity to expandable/compressible chambers [1697 for the default (or waste) side and 1699 for the sort side] adjacent to the flowcell inner wall and separated from the sheath fluid 1620 by a flexible membrane (1696 for the default side and 1698 for the sort side). In the default state, depicted in FIG. 16A, the expandable chambers 1697 and 1699 of both the default-side and sort-side actuators are in their default configuration at pressures designed to match the pressure of the fluid inside the flowcell at the location of the membranes 1696 and 1698, resulting in flat shapes of the membranes to match the shape of the flowcell inner walls. In this non-sorting state, a non-selected particle 1650 in the sample core stream 1630 flows by design into the default (or waste) sorting channel 1646. In the sorting state, depicted in FIG. 16B, the expandable chamber 1697 of the default-side actuator 1680 is pressurized (by means including, without limitation, heating means, mechanical means, hydraulic means, and/or gas injection means), through actuation, in a similar way as depicted in reference to FIG. 15B; this pressure differential with respect to the local pressure in the sheath fluid causes the membrane 1696 to bulge into the flowcell until a new equilibrium is reached. Simultaneously, the compressible chamber 1699 of the sorting side actuator 1681 is depressurized (by means including, without limitation, cooling means, mechanical means, hydraulic means, and/or gas aspiration means), through activation of actuator 1681, to a lower pressure than in the default configuration; this pressure differential with respect to the local pressure in the sheath fluid causes the membrane 1698 to flex away from the flowcell until a new equilibrium is reached. The combination of the inwardly bulging default-side membrane 1696 and the outwardly flexing sort-side membrane 1698 causes the flow pattern to shift in a similar way to that previously shown for FIGS. 14B and 15B, resulting in the sample core stream 1631 being temporarily diverted into the sorting channel 1648, which sample stream contains a particle 1655 detected upstream and automatically selected by analysis circuitry and/or algorithms to trigger sorting actuation. Following deactivation of the actuator pair, both the default-side and the sort-side expandable/compressible chambers 1697 and 1699 are allowed to or made to return to their default pressure states, both the default-side and the sort-side membranes 1696 and 1698 return to their default flat shapes, and the flow pattern returns to the original default configuration of FIG. 16A.

FIGS. 17A-17D illustrate a multi-way sorting embodiment of a particle analysis and sorting method of the current disclosure. Each of the four figures shows a schematic representation of a cross-sectional view of the sorting region of the flowcell. The configuration is similar to that depicted in reference to FIGS. 14A and 14B, except that instead of a single sorting channel, a plurality of sorting channels 1741-1744 is provided along a transverse direction y. One advantage of this embodiment is the ability to have a plurality of different receptacles into which the sample may be sorted, depending on the result of the upstream analysis by the interrogating light beam, the signal detectors, and associated electronic and logic trigger circuitry and/or algorithms. For example, the signals detected in response to the upstream interrogation of the sample may indicate that a particle, e.g., particle 1751, was detected with a certain set A of properties targeted for selection (e.g., the presence of surface antigens or intracellular markers associated with certain kinds of cancer cells). It may be desirable to sort particles having these properties into a certain collection receptacle, e.g., one provided to receive the outflow from sorting channel 1741, as illustrated in FIG. 17B. Another particle, e.g., particle 1752, may flow through the interrogation point and produce signals that indicate the presence of a different set B of properties targeted for selection (e.g., the presence of surface antigens or intracellular markers associated with certain kinds of stem cells). It would be desirable to sort particles like particle 1752 having set-B properties into a different receptacle from that designed for collection of particles having set-A properties: e.g., a receptacle provided to receive the outflow from sorting channel 1742, as illustrated in FIG. 17C. Likewise for yet another set D of properties, particles like particle 1754 detected as having those properties, and a sorting channel 1744 designed to flow into a receptacle to collect such particles. Accordingly, the embodiment illustrated in FIGS. 17A-17D provides an example of such a multi-way sorting capability of the current disclosure, with a number of sorting channels 1741-1744 in addition to the default (or waste) sorting channel 1746. FIGS. 17A-17D exemplarily show four such sorting channels explicitly. It will be apparent to those skilled in the art that additional configurations having more or less than four sorting channels, in addition to the default sorting channel, do not depart from the scope of the current disclosure.

Each of the sorting channels 1741-1744 (as well as the default sorting channel 1746) may optionally be connected with a receiving receptacle designed to collect the fluid flow from the respective channel. The selection of a particular sorting channel (or of the default sorting channel) as the target for reception of a desired sorted portion of the sample core stream is effected by actuation of actuator 1780. In a two-way sort there are two principal sorting states, which can be described as OFF (default) and ON (sorting) as described above in relation to FIGS. 14A-14B, 15A-15B, and 16A-16B. In a multi-way sort, on the other hand, there generally can be as many sorting states as there are sorting “ways” or possible sorting channels. With reference to FIGS. 17A-17D, five possible sorting channels are indicated (the default sorting channel 1746 plus four sorting channels 1741-1744); accordingly, this is referred to as a five-way sort (or, alternatively, as a four-way sort, the default/waste sorting channel being excluded from the count in such case). An actuation process is provided to result in different degrees of deflection of the sample core stream portion, corresponding to the selection of different intended sorting channels.

In FIG. 17A actuator 1780 is depicted as embodied in, in contact with, or in proximity of the inner wall of the flowcell 1700 on the default sorting channel side. Similarly to the state depicted in FIG. 14A, FIG. 17A shows the configuration of the default state, where with the actuator 1780 in the OFF state, a non-selected particle 1750 in the sample core stream 1730 flows by design into the default sorting channel 1746. Similarly to the state depicted in FIG. 14B, FIGS. 17B-17D show the configurations of various sorting states, where with the actuator 1780 in the ON state at levels 1, 2, and 4, respectively, transient regions 1791, 1792, and 1794, respectively (comprising, without limitation, a gas, vapor, gas-vapor bubble, or a less-dense region of sheath fluid), are generated (by means including, without limitation, thermal means, electrolytic means, and gas injection means), which create respective localized flow diversions in the depicted cross-sectional plane and in its vicinity, which diversions temporarily deflect the sample core stream into configurations 1731, 1732, and 1734, respectively, and cause the corresponding particles 1751, 1752, and 1754, respectively, to flow into the respective sorting channels 1741, 1742, and 1744. Following deactivation of the actuator, the transient gas bubble shrinks or is cleared away, and the flow pattern returns to the original default state of FIG. 17A. Not shown is the configuration of a sorting state intermediate to the sorting states of FIGS. 17C and 17D, corresponding to an actuation level 3, whereby a transient region of a size intermediate between that of regions 1792 and 1794 temporarily diverts the sample core stream into sorting channel 1743.

Throughout this disclosure the term “default sorting channel” (or “waste sorting channel”) is associated with an OFF state of an actuator, signifying a passive state in which no particle sorting is performed, and in which the sample core stream and particles therein are typically outflowed and discarded as undesired waste. The term “sorting channel” is associated with an ON state of an actuator, signifying an activated state of an actuator, in which active sorting of a desired particle is performed. While for some embodiments this may be a preferred configuration, the current disclosure is not so limited, and included under the scope of the disclosure are embodiments where a passive state of an actuator is associated with collection of desired particles, and an active state of an actuator is associated with generation of a waste stream of undesired particles from the particle analyzer/sorter.

Referring to FIG. 18B, a flow chart is provided that describes a sequence of principal steps involved in the performance of a method of particle analysis in accordance with an embodiment of the present disclosure. A first step 1840 involves the generation of one or more trains of optical pulses (or other modulated output of light from one or more sources) for optical interrogation of particles in a sample. A second step 1842 involves the presentation of a sample, or the introduction of a sample, to the apparatus by a user or operator. A third, optional, step 1844 involves the mixing, reaction, and incubation of the sample with one or more reagents, which reagents may be preloaded onboard the apparatus or may be introduced by the user or operator. A fourth step 1846 involves the formation, by means of, e.g., hydrodynamic focusing of the sample by sheath fluid, of a core stream of particles flowing essentially in single file in the microchannel portion of the flowcell for optical interrogation. A fifth step 1848 involves the interrogation, by optical interaction, of a single particle in the sample core stream by the one or more trains of optical pulses, resulting in the generation of optical interaction signals. A sixth step 1850 involves the collection of the optical interaction signals, the optical filtering of the collected optical signals, and the spectral isolation of the filtered optical signals. A seventh step 1852 involves the detection of the spectrally isolated optical signals, the transduction of said signals into analog electrical signals, the digitization of the analog electrical signals into digital signals (also referred to as digitizations, digitization points, digital samplings, or sampling points), and the processing of the digital signals. An eighth step 1854 involves the further application of digital signal processing algorithms to the digital signals corresponding to each isolated spectral band so as to isolate the separate contributions of one or more fluorescence lifetime components to each signal. A ninth optional step 1856 involves the coherent summing, or coherent averaging, of lifetime signals coming from different pulses but all originating from the same particle under interrogation. A tenth step 1858 involves the recording and storage of the detected and processed signal parameters, including, without limitation, fluorescence intensity in one or more spectral bands, one or more fluorescence lifetime values in each of the one or more spectral bands, phase shift, scattering intensity, and absorption. An eleventh step 1860 involves a decision, which may be automated or may be presented by the system, through a processing unit, to the user or operator as a call for action, on whether to analyze additional particles; if the choice is positive, the method workflow returns to the fifth step above; if the choice is negative, the method workflow continues to the next (twelfth) step below. A twelfth optional step 1862 involves the classification of a portion or a totality of the events detected and analyzed according to certain criteria (which may include, without limitation, entities commonly referred to in the art as “triggers,” “thresholds,” and “gates”), which may be predetermined and preloaded into the apparatus or may be selected or modified or created by the user. A thirteenth step 1864 involves the presentation to the user or operator of the processed data (which may include, without limitation, the raw detected time-varying signals, a list of detected particle-interrogation events, and graphs or plots of detected events displayed according to characteristics such as, e.g., fluorescence lifetime, fluorescence intensity, absorption, and scattering) by means of a user interface such as, e.g., a screen, a computer monitor, a printout, or other such means.

FIG. 19 shows a block diagram of an exemplary embodiment of a data processing system 1900 to provide a particle analysis and sorting system as described herein. In an embodiment, data processing system 1900 is a part of the control system to perform a method that includes providing light pulses; providing a sample for analysis; exposing the sample to the light pulses; detecting optical decay curves; and extracting time constants from the optical decay curves, as described herein. In some embodiments, data processing system 1900 is represented by any one of signal processing units 790, 890, 990 and 1090 depicted in FIGS. 7, 8, 9, and 10 , respectively, and further optionally incorporates any one of data storage units 792 and 892 depicted in FIGS. 7 and 8 , respectively.

Data processing system 1900 includes a processing unit 1901 that may include a microprocessor or microcontroller, such as Intel microprocessor (e.g., Core i7, Core 2 Duo, Core 2 Quad, Atom), Sun Microsystems microprocessor (e.g., SPARC), IBM microprocessor (e.g., IBM 750), Motorola microprocessor (e.g., Motorola 68000), Advanced Micro Devices (“AMD”) microprocessor, Texas Instrument microcontroller, and any other microprocessor or microcontroller.

Processing unit 1901 may include a personal computer (PC), such as a Macintosh® (from Apple Inc. of Cupertino, Calif.), Windows®-based PC (from Microsoft Corporation of Redmond, Wash.), or one of a wide variety of hardware platforms that run the UNIX operating system or other operating systems. For at least some embodiments, processing unit 1901 includes a general purpose or specific purpose data processing system based on Intel, AMD, Motorola, IBM, Sun Microsystems, IBM processor families, or any other processor families. As shown in FIG. 19 , a memory 1903 is coupled to the processing unit 1901 by a bus 1923. Memory 1903 has instructions and data 1904 stored thereon which when accessed by processing unit 1901 cause the processing unit 1901 to perform methods to provide highly multiplexed particle analysis or lifetime analysis, and optionally sorting, as described herein. Memory 1903 can be dynamic random access memory (“DRAM”) and can also include static random access memory (“SRAM”). A bus 1923 couples processing unit 1901 to memory 1903 and also to a non-volatile storage 1909 and to a display controller 1905 (if a display is used) and to input/output (I/O) controller(s) 1911. Display controller 1905 controls in the conventional manner a display on a display device 1907 which can be a cathode ray tube (CRT), a liquid crystal display (LCD), a light-emitting diode (LED) monitor, a plasma monitor, or any other display device. Input/output devices 1917 can include a keyboard, disk drives, printers, a scanner, a camera, and other input and output devices, including a mouse or other pointing device. I/O controller 1911 is coupled to one or more audio input devices 1913 such as, for example, one or more microphones.

Display controller 1905 and I/O controller 1911 can be implemented with conventional well-known technology. An audio output 1915 such as, for example, one or more speakers, may be coupled to I/O controller 1911. Non-volatile storage 1909 is often a magnetic hard disk, an optical disk, or another form of storage for large amounts of data. Some of this data is often written, by a direct memory access process, into memory 1903 during execution of software in data processing system 1900 to perform methods described herein.

One skilled in the art will immediately recognize that the terms “computer-readable medium” and “machine-readable medium” include any type of storage device that is accessible by processing unit 1901. Data processing system 1900 can interface to external systems through a modem or network interface 1921. It will be appreciated that modem or network interface 1921 can be considered to be part of data processing system 1900. This interface 1921 can be an analog modem, ISDN modem, cable modem, token ring interface, satellite transmission interface, Wi-Fi, Bluetooth, cellular network communication interface, or other interfaces for coupling a data processing system to other data processing systems.

It will be appreciated that data processing system 1900 is one example of many possible data processing systems which have different architectures. For example, personal computers based on an Intel microprocessor often have multiple buses, one of which can be an input/output (I/O) bus for the peripherals and one that directly connects processing unit 1901 and memory 1903 (often referred to as a memory bus). The buses are connected together through bridge components that perform any appropriate translation due to differing bus protocols.

Network computers are another type of data processing system that can be used with the embodiments as described herein. Network computers do not usually include a hard disk or other mass storage, and the executable programs are loaded from a network connection into memory 1903 for execution by processing unit 1901. A typical data processing system will usually include at least a processor, memory, and a bus coupling the memory to the processor.

It will also be appreciated that data processing system 1900 can be controlled by operating system software which includes a file management system, such as a disk operating system, which is part of the operating system software. Operating system software can be the family of operating systems known as Macintosh® Operating System (Mac OS®) or Mac OS X® from Apple Inc. of Cupertino, Calif., or the family of operating systems known as Windows® from Microsoft Corporation of Redmond, Wash., and their associated file management systems. The file management system is typically stored in non-volatile storage 1909 and causes processing unit 1901 to execute the various acts required by the operating system to input and output data and to store data in memory, including storing files on non-volatile storage 1909.

In various embodiments, hardwired circuitry may be used in combination with software instructions to implement methods described herein. A non-transitory machine-readable medium can be used to store software and data which when executed by a data processing system causes the system to perform various methods described herein. This executable software and data may be stored in various places including for example ROM, volatile RAM, non-volatile memory, and/or cache. Portions of this software and/or data may be stored in any one of these storage devices.

Thus, a machine-readable medium includes any mechanism that provides (i.e., stores and/or transmits) information in a form accessible by a machine (e.g., a computer, network device, or any device with a set of one or more processors, etc.). For example, a machine-readable medium includes recordable/non-recordable media [e.g., read only memory (ROM); random access memory (RAM); magnetic disk storage media; optical storage media; flash memory devices; and the like].

It will be further appreciated that data processing system 1900 may be functionally implemented by allocating several of its functions to distributed units or modules separate from a central system. In some embodiments, some or all of the signal processing functions as depicted, e.g., in FIGS. 7-10 , illustrated in FIGS. 11A-11F and 12A-12B, and described in FIGS. 18A-18C, may be performed by signal processing units or modules physically separate from data processing system 1900, yet connected with it for performance of other functions, such as, e.g., input/output, display, data storage, memory usage, bus usage, additional signal processing functions, and both specific-purpose and general-purpose data processing functions. In some embodiments, some or all of the data storage functions as depicted, e.g., in FIGS. 7-8 , illustrated in FIGS. 11A-11F and 12A-12B, and described in FIGS. 18A-18C, may be performed by data storage units or modules physically separate from data processing system 1900, yet connected with it as described above. In some embodiments, some or all of the signal processing functions mentioned may be performed by processing unit 1901 internal to data processing system 1900, and in some embodiments some or all of the data storage functions mentioned may be performed by non-volatile storage unit 1909 and/or memory unit 1903 internal to data processing system 1900.

The methods as described herein can be implemented using dedicated hardware (e.g., using Field Programmable Gate Arrays, Digital Signal Processing chips, or Application Specific Integrated Circuits) or shared circuitry (e.g., microprocessors, microcontrollers, single-board computers, standalone computers, or cloud-based processors on remote servers) under control of program instructions stored in a machine-readable medium. The methods as described herein can also be implemented as computer instructions for execution on a data processing system, such as system 1900 of FIG. 19 .

It will be appreciated by those skilled in the art that aspects of the present disclosure, while illustrated with reference to applications to particle analysis and sorting and particularly to flow cytometry, also present advantages in other application areas. The concept of lifetime binning as a means to simplify the performance of lifetime measurements and thereby enable higher degree of multiplexing than hitherto practical, for example, is also advantageous to the field of imaging, in particular to the field of microscopy, and more particularly to the field of fluorescence microscopy, including confocal scanning microscopy. Whereas in a flow cytometer an “event” is defined as the passage of a particle through the interrogation area, in microscopy the roughly equivalent element is a “pixel” (or “voxel” in certain applications of confocal scanning microscopy), defined as the smallest resolvable unit of an image (or of a three-dimensional measured volume in certain applications of confocal scanning microscopy). Spectral spillover and crosstalk is a problem in fluorescence microscopy just as it is a problem in flow cytometry, and the present disclosure offers a solution to both by providing a greater degree of multiplexing, a reduced level of spectral spillover, a reduced interference from autofluorescence, or a combination of the three. The present disclosure admits of implementation within the framework of a fluorescence microscope in ways that parallel very closely the specific examples given in the case of flow-based particle analysis. A microscopy application of the present disclosure, for example, would rely on a system configuration very similar to that of FIG. 7 , with the fluid elements 740, 742, 700, and 740 replaced by a suitable sample holder, such as are well known in the art, for exposure of the sample to the beam; and similarly for FIGS. 9 and 10 . In other words, adaptation of the present disclosure to the field of microscopy is fully within the scope of this disclosure, given the descriptions of the novel apparatus and methods herein. One specific application of fluorescence microscopy that would benefit from the present disclosure is in vivo imaging, such as, e.g., methods and apparatuses used for medical diagnostics. These include the analysis and diagnosis of externally optically accessible organs, such as the skin and the eye, as well as organs optically accessible through the use of endoscopes, such as the respiratory tract, the gastrointestinal tract, and, in the context of surgery, any other organ or part of the body. As in the case of laboratory-based fluorescence microscopy, adaptation of the apparatuses and methods described herein is entirely within the scope of the present disclosure, requiring only minor modifications of the apparatus and process steps from the illustrative examples that are provided. The usefulness of the present disclosure is therefore not to be circumscribed to the examples and figures provided, but extends to the full scope of what is claimed.

It will be further appreciated by those skilled in the art that the concept of lifetime binning as a means to enable higher degree of multiplexing than hitherto possible or practical, for example, is also advantageous to the field of bead-based multiplexing assays. The various methods and systems described herein can be applied to the task of bead-based multiplexing with only minor modifications, such as accounting for the predesigned levels of each color-coding dye present in a bead in order to identify which combination of dye level(s) a particular bead belongs to. In an exemplary embodiment, microspheres (of, e.g., polystyrene or other materials) are formed so as to incorporate a dye having a relatively short fluorescence lifetime (e.g., dye A, having a lifetime shorter than, e.g., about 5 ns) and another dye having a relatively long fluorescence lifetime (e.g., dye B, having a lifetime longer than, e.g., about 10 ns), both such dyes having substantially overlapping spectra so as to permit their detection by the same optical apparatus. Each microsphere is prepared with one of a set of discrete amounts of each dye, e.g., an amount substantially or approximately equal to 1000, 1500, 2250, 3375, 5063, 7594, 11391, 17086, 25629, or 38443 molecules. In certain embodiments, more or less than the ten discrete amounts described in this exemplary embodiment may be preferable. In certain embodiments, the ratio of one amount to the immediately lower one may be preferably more or less than the ratio of 1.5 described in this exemplary embodiment. In certain embodiments, the ratios between amounts may preferably be other than the uniform ratio described in this exemplary embodiment. In certain embodiments, the lowest amount may preferably be more or less than the 1000 molecules described in this exemplary embodiment. The various combinations of the two dyes in different amounts comprise a multiplexing set; in this exemplary embodiment, the use of ten different amounts for each of two dyes yields 100 different combinations, each combination corresponding to a different type of coded microsphere. Each type of coded microsphere in turn is used for a different capture assay: e.g., beads having the combination of 1000 molecules of dye A and 1000 molecules of dye B are prepared with, e.g., capture antibodies specific to antigen X; beads having the combination of 1500 molecules of dye A and 1000 molecules of dye B are prepared with, e.g., capture antibodies specific to antigen Y; and so forth. The beads so prepared are then used in, e.g., multiplexing sandwich immunoassays, multiplexing nucleic-acid assays, or other multiplexing assays as described herein and according to methods well known to those skilled in the art.

An exemplary embodiment of an instrument used for bead-based multiplexing assays according to the present disclosure comprises: a pulsed light source designed to simultaneously excite dye A and dye B in each bead in a sample of beads suspended in buffer medium as the bead flows in single file through a flowcell; an optical apparatus designed to collect fluorescence emission decays from dye A and dye B; and a signal processing apparatus designed to extract the contributions from dye A and dye B to the observed decay signals; as well as a light source designed to excite one or more type of fluorescent reporter molecules; an optical apparatus designed to collect fluorescence emission from the reporter molecule(s) present on each detected bead; and a signal processing apparatus designed to measure the reporter fluorescence emission or emissions from each detected bead. The apparatus in this exemplary embodiment uses the detected levels of fluorescence in the bead-coding channel(s) to identify the specific dye combination that a detected bead belongs to, and therefore to determine what corresponding assay was performed on that bead. By allowing the simultaneous identification of each possible combination of dye amounts, and by incorporating fluorescence lifetime as an additional parameter for discrimination of bead-coding dyes, the apparatus and method of the present disclosure allows the multiplexing of assays in greater numbers than hitherto possible or practical. In certain embodiments, more than two dyes having overlapping spectra and having distinct lifetimes are used to code microspheres. In certain embodiments, more than one spectral channel of detection is allocated for the purpose of coding microspheres, each channel allowing the use of one or more dyes with overlapping spectra and distinct lifetimes. In certain embodiments, more than one light source is allocated for the purpose of exciting dyes used to code microspheres, and more than one optical apparatus is used to collect the light emitted by microsphere-coding dyes.

A method of analyzing particles in a sample using a particle analyzer is disclosed, comprising the steps of:

-   -   providing a source of a beam of pulsed optical energy;     -   providing a sample holder configured to expose a sample to the         beam;     -   providing a detector, the detector comprising a number of         spectral detection channels, the channels being sensitive to         distinct wavelength sections of the electromagnetic spectrum and         being configured to detect optical signals resulting from         interactions between the beam and the sample, the channels being         further configured to convert the optical signals into         respective electrical signals;     -   providing a first optical path from the source of the beam to         the sample;     -   providing a second optical path from the sample to the detector;     -   providing a signal processing module;     -   exposing the sample to the beam, and     -   using the signal processing module for:     -   receiving the electrical signals from the detector, wherein the         electrical signals represent a time-domain sequence of pulse         signals;     -   segmenting the sequence into equal pulse signal segments each         comprising substantially a same number of digitization elements,         wherein a length of each segment corresponds to an excitation         pulse repetition period;     -   coherently adding the individual pulse signal segments, based on         individual digitization elements, to form a supercurve, wherein         each digitization index of the supercurve corresponds to a value         equal to a sum of all the signal values from the corresponding         digitization indices from all pulse signal segments, and the         supercurve comprises at least two lifetime components, each         lifetime component having a slope that is inversely proportional         to a value of a corresponding time constant; and     -   quantifying an intensity contribution of a first short-lifetime         component and a second long-lifetime component relative to an         overall intensity of the supercurve.

A method of analyzing and sorting particles in a sample using a particle analyzer/sorter is disclosed, comprising the steps of:

-   -   providing an internally modulated laser as a source of a beam of         pulsed optical energy;     -   providing a flowcell configured as an optical excitation chamber         for exposing to the beam a sample comprising a suspension of         particles and for generating optical signals from interactions         between the beam and the particles;     -   providing a detector, the detector comprising a number of         spectral detection channels, the channels being sensitive to         distinct wavelength sections of the electromagnetic spectrum and         being configured to detect fluorescence optical signals         resulting from interactions between the beam and the particles         in the sample, the channels being further configured to convert         the optical signals into respective electrical signals;     -   providing a first optical path from the source of the beam to         the sample;     -   providing a second optical path from the sample to the detector;     -   providing a flow path for the suspension of particles;     -   providing connections between the flowcell and each of the flow         path, the first optical path, and the second optical path;     -   providing a signal processing module comprising one of an FPGA,         a DSP chip, an ASIC, a CPU, a microprocessor, a microcontroller,         a single-board computer, a standalone computer, and a         cloud-based processor;     -   exposing the particles in the sample to the beam;     -   using the signal processing module for:     -   receiving the electrical signals from the detector, wherein the         electrical signals represent a time-domain sequence of pulse         signals;     -   segmenting the sequence into equal pulse signal segments each         comprising substantially a same number of digitization elements,         wherein a length of each segment corresponds to an excitation         pulse repetition period;     -   coherently adding the individual pulse signal segments, based on         individual digitization elements, to form a supercurve, wherein         each digitization index of the supercurve corresponds to a value         equal to a sum of all the signal values from the corresponding         digitization indices from all pulse signal segments, and the         supercurve comprises at least two lifetime components, each         lifetime component having a slope that is inversely proportional         to a value of a corresponding time constant;     -   quantifying an intensity contribution of a first short-lifetime         component and a second long-lifetime component relative to an         overall intensity of the supercurve;     -   providing a particle sorting actuator connected with the flow         path, based on at least one flow diversion in the flow path, and         further based on one of a transient bubble, a pressurizable         chamber, a pressurizable/depressurizable chamber pair, and a         pressure transducer;     -   providing an actuator driver connected with the actuator, the         driver being configured to receive actuation signals from the         signal processing module;     -   providing at least one particle collection receptacle; and     -   collecting at least one particle from the suspension of         particles in the particle collection receptacle.

A method of analyzing particles in a sample using a particle analyzer is disclosed, comprising the steps of:

-   -   providing a source of a beam of pulsed optical energy;     -   providing a sample holder configured to expose a sample to the         beam;     -   providing a detector, the detector comprising a number of         spectral detection channels, the channels being sensitive to         distinct wavelength sections of the electromagnetic spectrum and         being configured to detect optical signals resulting from         interactions between the beam and the sample, the channels being         further configured to convert the optical signals into         respective electrical signals;     -   providing a first optical path from the source of the beam to         the sample;     -   providing a second optical path from the sample to the detector;     -   providing a signal processing module;     -   exposing the sample to the beam, and     -   using the signal processing module for:     -   receiving the electrical signals from the detector, wherein the         electrical signals represent a time-domain sequence of pulse         signals;     -   segmenting the sequence into equal pulse signal segments each         comprising substantially a same number of digitization elements,         wherein a length of each segment corresponds to an excitation         pulse repetition period;     -   coherently adding the individual pulse signal segments, based on         individual digitization elements, to form a supercurve, wherein         each digitization index of the supercurve corresponds to a value         equal to a sum of all the signal values from the corresponding         digitization indices from all pulse signal segments, and the         supercurve comprises at least two lifetime components, each         lifetime component having a slope that is inversely proportional         to a value of a corresponding time constant;     -   allocating each lifetime component of the supercurve to discrete         bins of predetermined time constants; and     -   quantifying an intensity contribution of a first short-lifetime         component and a second long-lifetime component relative to an         overall intensity of the supercurve based on the allocation of         the individual lifetime components to the discrete bins.

A method of analyzing and sorting particles in a sample using a particle analyzer/sorter is disclosed, comprising the steps of:

-   -   providing an internally modulated laser as a source of a beam of         pulsed optical energy;     -   providing a flowcell configured as an optical excitation chamber         for exposing to the beam a sample comprising a suspension of         particles and for generating optical signals from interactions         between the beam and the particles;     -   providing a detector, the detector comprising a number of         spectral detection channels, the channels being sensitive to         distinct wavelength sections of the electromagnetic spectrum and         being configured to detect fluorescence optical signals         resulting from interactions between the beam and the particles         in the sample, the channels being further configured to convert         the optical signals into respective electrical signals;     -   providing a first optical path from the source of the beam to         the sample;     -   providing a second optical path from the sample to the detector;     -   providing a flow path for the suspension of particles;     -   providing connections between the flowcell and each of the flow         path, the first optical path, and the second optical path;     -   providing a signal processing module comprising one of an FPGA,         a DSP chip, an ASIC, a CPU, a microprocessor, a microcontroller,         a single-board computer, a standalone computer, and a         cloud-based processor;     -   exposing the particles in the sample to the beam;     -   using the signal processing module for:     -   receiving the electrical signals from the detector, wherein the         electrical signals represent a time-domain sequence of pulse         signals;     -   segmenting the sequence into equal pulse signal segments each         comprising substantially a same number of digitization elements,         wherein a length of each segment corresponds to an excitation         pulse repetition period;     -   coherently adding the individual pulse signal segments, based on         individual digitization elements, to form a supercurve, wherein         each digitization index of the supercurve corresponds to a value         equal to a sum of all the signal values from the corresponding         digitization indices from all pulse signal segments, and the         supercurve comprises at least two lifetime components, each         lifetime component having a slope that is inversely proportional         to a value of a corresponding time constant;     -   allocating each lifetime component of the supercurve to discrete         bins of predetermined time constants;     -   quantifying an intensity contribution of a first short-lifetime         component and a second long-lifetime component relative to an         overall intensity of the supercurve based on the allocation of         the individual lifetime components to the discrete bins;     -   providing a particle sorting actuator connected with the flow         path, based on at least one flow diversion in the flow path, and         further based on one of a transient bubble, a pressurizable         chamber, a pressurizable/depressurizable chamber pair, and a         pressure transducer;     -   providing an actuator driver connected with the actuator, the         driver being configured to receive actuation signals from the         signal processing module;     -   providing at least one particle collection receptacle; and     -   collecting at least one particle from the suspension of         particles in the particle collection receptacle.

In the foregoing specification, embodiments of the current disclosure have been described with reference to specific exemplary embodiments thereof. It will, however, be evident that various modifications and changes may be made thereto without departing from the broader spirit and scope of the disclosure. The specification and drawings are, accordingly, to be regarded in an illustrative rather than a restrictive sense. 

The invention claimed is:
 1. An apparatus for analyzing an optical signal decay, comprising: a source of a beam of pulsed optical energy configured to expose a sample to the beam; a detector comprising a number of spectral detection channels sensitive to distinct wavelength sections of the electromagnetic spectrum, wherein the channels are configured to detect optical signals resulting from interactions between the beam and the sample and convert the optical signals into respective electrical signals; a first optical path from the source of the beam to the sample; a second optical path from the sample to the detector; and a signal processing module configured to execute a lifetime analysis algorithm comprising: receiving the electrical signals from the detector, wherein the electrical signals represent a time-domain sequence of pulse signals; segmenting the sequence into equal pulse signal segments each comprising substantially a same number of sampling points, wherein each sampling point of each pulse signal segment corresponds to a respective sampling index, and wherein a length of each pulse signal segment corresponds to an excitation pulse repetition period; coherently adding a value of a sampling point corresponding to the respective sampling index of each pulse signal segment to form a supercurve, wherein each sampling index of the supercurve corresponds to a value equal to a sum of substantially all the sampling point values from the corresponding sampling indices from each pulse signal segment, and wherein the supercurve comprises an intensity of at least one lifetime component over time; determining whether the at least one lifetime component of the supercurve comprises one or both of: a short-lifetime component and a long-lifetime component; and quantifying an intensity contribution of the at least one lifetime component of the supercurve.
 2. The apparatus of claim 1, wherein the optical signals comprise a fluorescence signal.
 3. The apparatus of claim 2, wherein, when present, the short-lifetime component comprises autofluorescence and the long-lifetime component comprises exogenous fluorescence.
 4. The apparatus of claim 1, wherein the at least one lifetime component comprises an instrument response function when the short-lifetime component and the long-lifetime component are not present.
 5. The apparatus of claim 1, wherein the sample comprises a suspension of particles, such that the apparatus further comprises: a flow path for the suspension of particles; and a flowcell configured as an optical excitation chamber for generating the optical signals from interactions between the beam of pulsed optical energy and the particles, wherein the flowcell is connected with the flow path, the first optical path, and the second optical path.
 6. The apparatus of claim 5, further comprising: a particle sorting actuator connected with the flow path; an actuator driver connected with the actuator, wherein the driver is configured to receive actuation signals from the signal processing module; and at least one particle collection receptacle connected with the flow path.
 7. The apparatus of claim 6, wherein the particle sorting actuator is based on at least one flow diversion in the flow path.
 8. The apparatus of claim 7, wherein the particle sorting actuator is based on one of: a transient bubble, a pressurizable chamber, a pressurizable/depressurizable chamber pair, or a pressure transducer.
 9. The apparatus of claim 1, wherein the signal processing module is further configured to extract time constants from the supercurve.
 10. The apparatus of claim 9, wherein the signal processing module is further configured to allocate each lifetime component of the supercurve to predetermined bins, each bin representing a group of relatively similar lifetime components.
 11. An apparatus for analyzing an optical signal decay, comprising: a source of a beam of pulsed optical energy configured to expose a sample to the beam; a detector comprising a number of spectral detection channels sensitive to distinct wavelength sections of the electromagnetic spectrum, wherein the channels are configured to detect optical signals resulting from interactions between the beam and the sample and convert the optical signals into respective electrical signals; a first optical path from the source of the beam to the sample; a second optical path from the sample to the detector; and a signal processing module configured to execute a lifetime analysis algorithm comprising: receiving the electrical signals from the detector, wherein the electrical signals represent a time-domain sequence of pulse signals; segmenting the sequence into equal pulse signal segments each comprising substantially a same number of sampling points, wherein each sampling point of each pulse signal segment corresponds to a respective sampling index, and wherein a length of each pulse signal segment corresponds to an excitation pulse repetition period; coherently adding a value of a sampling point corresponding to the respective sampling index of each pulse signal segment to form a supercurve, wherein each sampling index of the supercurve corresponds to a value equal to a sum of substantially all the sampling point values from the corresponding sampling indices from each pulse signal segment, and wherein the supercurve comprises an intensity of at least one lifetime component over time; adding all the intensity values corresponding to each sampling index of the supercurve to generate a sum; normalizing the sum by dividing the sum by a peak value of the supercurve; determining a relative intensity contribution of each lifetime component by comparing the normalized sum to a lookup table; and determining an absolute intensity contribution of each lifetime component by multiplying the relative intensity contribution by the peak value of the supercurve.
 12. The apparatus of claim 11, wherein the signal processing module is further configured to subtract a baseline value from each respective sampling point.
 13. The apparatus of claim 11, wherein the signal processing module is further configured to measure a baseline shift produced by the at least one lifetime component as compared to a baseline when the at least one lifetime component is not present.
 14. The apparatus of claim 13, wherein the signal processing module is further configured to subtract the baseline shift from the received electrical signals.
 15. An apparatus for analyzing an optical signal decay, comprising: a source of a beam of pulsed optical energy configured to expose a sample to the beam, wherein the sample comprises one or more fluorophores having at least one lifetime component; a detector comprising a number of spectral detection channels sensitive to distinct wavelength sections of the electromagnetic spectrum, wherein the channels are configured to detect optical signals resulting from interactions between the beam and the sample and convert the optical signals into respective electrical signals; a first optical path from the source of the beam to the sample; a second optical path from the sample to the detector; and a signal processing module configured to execute a lifetime analysis algorithm comprising: receiving the electrical signals from the detector, wherein the electrical signals represent a time-domain sequence of pulse signals; segmenting the sequence into equal pulse signal segments each comprising substantially a same number of sampling points, wherein a length of each segment corresponds to an excitation pulse repetition period; measuring a peak baseline shift of the electrical signals based on a presence of the at least one lifetime component; and calculating an intensity contribution of the at least one lifetime component of the sample based on the measured peak baseline shift and one of: comparison to a lookup table and numerical fitting.
 16. The apparatus of claim 15, wherein the optical signals comprise a fluorescence signal.
 17. The apparatus of claim 16, wherein the at least one lifetime component is longer than the excitation pulse repetition period and comprises exogenous fluorescence.
 18. The apparatus of claim 15, wherein the signal processing module is further configured to subtract the baseline shift from the received electrical signals.
 19. The apparatus of claim 18, wherein the signal processing module is further configured to: coherently add a value of a sampling point corresponding to a respective sampling index of each pulse signal segment to form a supercurve, wherein each sampling index of the supercurve corresponds to a value equal to a sum of substantially all the sampling point values from the corresponding sampling indices from each pulse signal segment, and wherein the supercurve comprises an intensity of the at least one lifetime component over time; and quantifying an intensity contribution of the at least one lifetime component of the supercurve.
 20. The apparatus of claim 15, wherein the sample comprises a suspension of particles, such that the apparatus further comprises: a flow path for the suspension of particles; and a flowcell configured as an optical excitation chamber for generating the optical signals from interactions between the beam of pulsed optical energy and the particles, wherein the flowcell is connected with the flow path, the first optical path, and the second optical path. 